Porous beta-tricalcium phosphate granules for regeneration of bone tissue

ABSTRACT

A porous β-tricalcium phosphate material for bone implantation is provided. The multiple pores in the porous TCP body are separate discrete voids and are not interconnected. The pore size diameter is in the range of 20-500 μm, preferably 50-125 μm. The porous β-TCP material provides a carrier matrix for bioactive agents and can form a moldable putty composition upon the addition of a binder. Preferably, the bioactive agent is encapsulated in a biodegradable agent. The invention provides a kit and an implant device comprising the porous β-TCP, and a bioactive agent and a binder. The invention also provides an implantable prosthetic device comprising a prosthetic implant having a surface region, a porous β-TCP material disposed on the surface region and optionally comprising at least a bioactive agent or a binder. Methods of producing the porous β-TCP material and inducing bone formation are also provided.

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 09/798,518, filed Mar. 2, 2001.

BACKGROUND OF THE INVENTION

Bone tissue in the human body comprises the largest proportion of thebody's connective tissue mass. However, unlike other connective tissues,its matrix consists of physiologically mineralized, tiny crystallites ofa basic, carbonate-containing calcium phosphate called hydroxyapatitedistributed in an organized collagen structure. Repair of this tissue isa complex process involving a number of cellular functions directedtowards the formation of a scaffold and mineralization of the defectfollowed by an eventual remodeling of the defect site to attain theoriginal structure.

Implantations of calcium phosphate based biomaterials have been found tobe generally compatible and conducive to bone repair. Bone repair isinfluenced by a number of physico-chemical variables associated withcalcium phosphate such as the calcium to phosphate molar ratio.Hydroxyapatite and tricalcium phosphate are widely used in boneimplants. Hydroxyapatite has the chemical formula Ca₁₀(PO₄)₆(OH)₂, andthe ratio of calcium to phosphate is about 1.67. Tricalcium phosphate(TCP) has the formula of Ca₃(PO₄)₂, and the ratio of calcium tophosphate is about 1.5. Tricalcium phosphate has biological propertiesof being non-reactive and resorbable. It acts as a scaffolding for boneingrowth and undergoes progressive degradation and replacement by bone(Lange et al., Annals of Clinical and Laboratory Science, 16, pp.467-472 (1986)). TCP is degraded 10-20 times faster than hydroxyapatite.A TCP implant generally results in superior remodeling thanhydroxyapatite during the final stage of bone formation. It isnoteworthy that TCP is resorbed by osteoclast cells, whereas, the muchslower resorption of hydroxyapatite is effected mainly by foreign-bodygiant cells. The giant cells have a limit as to the amount ofhydroxyapatite they will resorb.

Porous ceramic material is often selected as the matrix for boneimplants. When such material is embedded at the implant site, the porousmaterial is resorbed by osteolytic cells which infiltrate the pores.Simultaneously, the bone tissue is regenerated by osteoblasts. A certainpore size is required for osteoblasts to invade the pore of the implantmaterial. Parameters such as crystallinity, solubility, particle size,porosity, pore structure and pore size of the implanted material cangreatly influence bone compatibility and bone integration. Aninappropriate combination of the above parameters can lead to improperbone repair.

The use of porous ceramics having interconnected pores as an implantablesolid material for bone substitutes has been described (see, e.g., U.S.Pat. No. 5,171,720; see also Frayssinet et al., Biomaterials, 14, pp.423-429 (1993)). Such porous ceramics, however, are brittle and are notcapable of being easily shaped by the practitioner during an operation.

Excessively large pore size and high porosity of the ceramic materialcan lead to excessive resorption rates, thus, preventing the matrix fromproviding a scaffold for the newly synthesized bone. When the rate ofresorption is faster than the rate of bone growth, it often leads to aninflammatory response. Small pore size and low porosity of the ceramicmaterial will lead to low resorption rates causing encapsulation ofmatrix particles in the new bone.

It would thus be desirable to identify a biomaterial which can beapplied to a defect site and which can greatly enhance the regenerativeprocess, particularly when used with other bioactive agents such as bonemorphogenic proteins and other related factors. In addition, it would bedesirable to identify and use a matrix which acts as a mechanicallydurable carrier for the bioactive agents and is a well-tolerated bonereplacement material that favors healing.

SUMMARY OF THE INVENTION

The present invention solves these problems by identifying a porousceramic material having a composition, pore size, porosity and granulesize for improving the regeneration of bone tissue in a living body, andrepairing a bone defect in a human or animal. The present inventionprovides a porous β-tricalcium phosphate (β-TCP) material for use inbone implant applications. The invention provides porous forms of β-TCPgranules which are biocompatible and support the development of new bonethroughout its structural form.

The invention also provides a composition comprising the porous β-TCPwith a bioactive agent such as an antibiotic, a bone morphogenic protein(BMP), or a nucleic acid molecule comprising a sequence encoding BMP inthe presence or absence of a morphogenic protein stimulatory factor(MPSF) to improve osteoconductivity. In a preferred embodiment, thebioactive agent is encapsulated in a biodegradable agent. Preferably,the particle size of the biodegradable agent is 20-500 μm. The porousβ-TCP material or porous β-TCP/bioactive agent mixture can also be usedin conjunction with binders to form a moldable putty composition readyfor shaping in the implant site. The invention also provides a kitcomprising the porous β-TCP, and at least one or more additionalcomponents including a bioactive agent and a binder.

In another aspect, the invention also provides an implantable devicecomprising the porous β-TCP material, and optionally comprising one ormore additional components including a bioactive agent such as a BMP, anantibiotic or a binder. The invention also provides an implantableprosthetic device comprising the porous β-TCP material and optionallycomprising one or more additional components including a bioactive agentsuch as a BMP, an antibiotic or a binder. The prosthetic device orimplantable device comprising the porous β-TCP and BMP may optionallycomprise a MPSF.

Another object of the invention is to provide a method of producing theporous β-TCP material. The method comprises blending the TCP powder witha pore-forming agent, adding a granulating solution to form a crumblymass, passing the crumbly mass through a sieve to form granules andsintering the granules to form the porous β-TCP.

The invention also provides a method of inducing bone formation in amammal comprising the step of implanting in the defect site of a mammala composition comprising the porous β-TCP and optionally a binder and/ora bioactive agent. The invention describes a method of delivering abioactive agent at a site requiring bone formation comprising implantingat the defect site of a mammal a composition comprising the porous β-TCPand a bioactive agent, wherein the bioactive agent is optionallyencapsulated in a biodegradable agent. The invention also describes amethod of delivering a bioactive agent to a site requiring cartilageformation comprising implanting at the defect site of a mammal acomposition comprising the bioactive agent and biodegradable agent,wherein the bioactive agent is encapsulated in the biodegradable agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Histologic image of animal number 297L (left tibia) at 4 weekswith placebo. From top to bottom, the sites are proximal, middle anddistal, each containing β-TCP putty 89B, β-TCP putty 89C, β-TCP putty89F, respectively.

FIG. 2. Histologic image of animal number 297R (right tibia) at 4 weekswith placebo. From top to bottom, the sites are proximal, middle anddistal, each containing control, collagen 48C, β-TCP putty 89A,respectively.

FIG. 3. Histologic image of animal number 295L (left tibia) at 4 weekswith OP-1. From top to bottom, the sites are proximal, middle anddistal, each containing collagen 48C, β-TCP putty 89A, β-TCP putty 89B,respectively.

FIG. 4. Histologic image of animal number 295R (right tibia) at 4 weekswith OP-1. From top to bottom, the sites are proximal, middle anddistal, each containing β-TCP putty 89C, β-TCP putty 89F, control,respectively.

FIG. 5. Histologic image of animal number 299L (left tibia) at 8 weekswith placebo. From top to bottom, the sites are proximal, middle anddistal, each containing β-TCP putty 89B, β-TCP putty 89C, β-TCP putty89F, respectively.

FIG. 6. Histologic image of animal number 299R (right tibia) at 8 weekswith placebo. From top to bottom, the sites are proximal, middle anddistal, each containing control, collagen 48C, β-TCP putty 89A,respectively.

FIG. 7. Histologic image of animal number 138L (left tibia) at 8 weekswith OP-1. From top to bottom, the sites are proximal, middle anddistal, each containing β-TCP putty 89A, β-TCP putty 89B, β-TCP putty89C, respectively.

FIG. 8. Histologic image of animal number 138R (right tibia) at 8 weekswith OP-1. From top to bottom, the sites are proximal, middle anddistal, each containing β-TCP putty 89F, control, collagen 48C,respectively.

FIG. 9. Radiographic image of animal number 297L (left tibia) at 4 weekswith placebo. From the right, the sites are proximal, middle and distal,each containing β-TCP putty 89B, β-TCP putty 89C, β-TCP putty 89F,respectively.

FIG. 10. Radiographic image of animal number 297R (right tibia) at 4weeks with placebo. From the left, the sites are proximal, middle anddistal, each containing control, collagen 48C, β-TCP putty 89A,respectively.

FIG. 11. Radiographic image of animal number 295L (left tibia) at 4weeks with OP-1. From the left, the sites are proximal, middle anddistal, each containing collagen 48C, β-TCP putty 89A, β-TCP putty 89B,respectively.

FIG. 12. Radiographic image of animal number 295R (right tibia) at 4weeks with OP-1. From the left, the sites are proximal, middle anddistal, each containing β-TCP putty 89C, β-TCP putty 89F, control,respectively.

FIG. 13. Radiographic image of animal number 299L (left tibia) at 8weeks with placebo. From the right, the sites are proximal, middle anddistal, each containing β-TCP putty 89B, β-TCP putty 89C, β-TCP putty89F, respectively.

FIG. 14. Radiographic image of animal number 299R (right tibia) at 8weeks with placebo. From the left, the sites are proximal, middle anddistal, each containing control, collagen 48C, β-TCP putty 89A,respectively.

FIG. 15. Radiographic image of animal number 138L (left tibia) at 8weeks with OP-1. From the right, the sites are proximal, middle anddistal, each containing β-TCP putty 89A, β-TCP putty 89B, β-TCP putty89C, respectively.

FIG. 16. Radiographic image of animal number 138R (right tibia) at 8weeks with OP-1. From the left, the sites are proximal, middle anddistal, each containing β-TCP putty 89F, control, collagen 48C,respectively.

FIG. 17. Paraffin scanning image of animal number 297L (left tibia) at 4weeks with placebo. From the top, the sites are proximal, middle anddistal, each containing β-TCP putty 89B, β-TCP putty 89C, β-TCP putty89F, respectively.

FIG. 18. Paraffin scanning image of animal number 297R (right tibia) at4 weeks with placebo. From the top, the sites are proximal, middle anddistal, each containing control, collagen 48C, β-TCP putty 89A,respectively.

FIG. 19. Paraffin scanning image of animal number 295L (left tibia) at 4weeks with OP-1. From the top, the sites are proximal, middle anddistal, each containing collagen 48C, β-TCP putty 89A, β-TCP putty 89B,respectively.

FIG. 20. Paraffin scanning image of animal number 295R (right tibia) at4 weeks with OP-1. From the top, the sites are proximal, middle anddistal, each containing β-TCP putty 89C, β-TCP putty 89F, control,respectively.

FIG. 21. Paraffin scanning image of animal number 299L (left tibia) at 8weeks with placebo. From the top, the sites are proximal, middle anddistal, each containing β-TCP putty 89B, β-TCP putty 89C, β-TCP putty89F, respectively.

FIG. 22. Paraffin scanning image of animal number 299R (right tibia) at8 weeks with placebo. From the top, the sites are middle and distal,each containing collagen 48C and β-TCP putty 89A, respectively.

FIG. 23. Paraffin scanning image of animal number 138L (left tibia) at 8weeks with OP-1. From the top, the sites are proximal, middle anddistal, each containing β-TCP putty 89A, β-TCP putty 89B, β-TCP putty89C, respectively.

FIG. 24. Paraffin scanning image of animal number 138R (right tibia) at8 weeks with OP-1. From the top, the sites are proximal, middle anddistal, each containing 5-TCP putty 89F, control, collagen 48C,respectively.

FIG. 25. Specimen 295L middle site showing one of the five pores withbone growth, where EP is an empty pore and FP is a filled pore.

FIG. 26. Specimen 299L distal site showing 7 or 8 pores with bonegrowth, where EP is any empty pore and FP is a filled pore.

FIG. 27. Radiographic image of animal number 5333L (left tibia) at 4weeks with OP-1 encapsulated in PLGA. From the left, the sites areproximal, middle and distal, each containing control, formulation 5 andformulation 4, respectively.

FIG. 28. Radiographic image of animal number 5335L (left tibia) at 8weeks with OP-1 encapsulated in PLGA. From the left, the sites areproximal, middle and distal, each containing control, formulation 4 andformulation 5, respectively.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention herein described may be fully understood,the following detailed description is set forth.

“Amino acid sequence homology” is understood to include both amino acidsequence identity and similarity. Homologous sequences share identicaland/or similar amino acid residues, where similar residues areconservative substitutions for, or “allowed point mutations” of,corresponding amino acid residues in an aligned reference sequence.Thus, a candidate polypeptide sequence that shares 70% amino acidhomology with a reference sequence is one in which any 70% of thealigned residues are either identical to, or are conservativesubstitutions of, the corresponding residues in a reference sequence.Certain particularly preferred morphogenic polypeptides share at least60%, and preferably 70% amino acid sequence identity with the C-terminal102-106 amino acids, defining the conserved seven-cysteine domain ofhuman OP-1, BMP-2, and related proteins.

Amino acid sequence homology can be determined by methods well known inthe art. For instance, to determine the percent homology of a candidateamino acid sequence to the sequence of the seven-cysteine domain, thetwo sequences are first aligned. The alignment can be made with, e.g.,the dynamic programming algorithm described in Needleman et al., J. Mol.Biol., 48, pp. 443 (1970), and the Align Program, a commercial softwarepackage produced by DNAstar, Inc. The teachings by both sources areincorporated by reference herein. An initial alignment can be refined bycomparison to a multi-sequence alignment of a family of relatedproteins. Once the alignment is made and refined, a percent homologyscore is calculated. The aligned amino acid residues of the twosequences are compared sequentially for their similarity to each other.Similarity factors include similar size, shape and electrical charge.One particularly preferred method of determining amino acid similaritiesis the PAM250 matrix described in Dayhoff et al., Atlas of ProteinSequence and Structure, 5, pp. 345-352 (1978 & Supp.), which isincorporated herein by reference. A similarity score is first calculatedas the sum of the aligned pairwise amino acid similarity scores.Insertions and deletions are ignored for the purposes of percenthomology and identity. Accordingly, gap penalties are not used in thiscalculation. The raw score is then normalized by dividing it by thegeometric mean of the scores of the candidate sequence and theseven-cysteine domain. The geometric mean is the square root of theproduct of these scores. The normalized raw score is the percenthomology.

“Biocompatible” refers to a material that does not elicit detrimentaleffects associated with the body's various protective systems, such ascell and humoral-associated immune responses, e.g., inflammatoryresponses and foreign body fibrotic responses. The term biocompatiblealso implies that no specific undesirable cytotoxic or systemic effectsare caused by the material when it is implanted into the patient.

“Binder” refers to any biocompatible material which, when admixed withosteogenic protein and/or the porous matrix promotes bone formation.Certain preferred binders promote such repair using less osteogenicprotein than standard osteogenic devices. Other preferred binders canpromote repair using the same amount of the osteogenic protein as thestandard osteogenic devices while some require more to promote repair.As taught herein, the skilled artisan can determine an effective amountof protein for use with any suitable binder using only routineexperimentation. Among the other characteristics of a preferred binderis an ability to render the device: pliable, shapeable and/or malleable;injectable; adherent to bone, cartilage, muscle and other tissues,resistant to disintegration upon washing and/or irrigating duringsurgery; and, resistant to dislodging during surgery, suturing andpost-operatively, to name but a few. Additionally, in certain preferredembodiments, a binder can achieve the aforementioned features andbenefits when present in low proportions.

“Biodegradable agent” refers to a resorbable biocompatible material suchas a material that degrades gradually at the implant site. The materialis capable of encapsulating a bioactive agent to provide time release orsustained release delivery of the bioactive agent. The biodegradablematerial encompasses natural and synthetic polymers. Examples ofbiodegradable material are poly(L-lactide) (PLLA), poly(D,L-lactide)(PDLLA), polyglycolide (PGA), poly(lactide-co-glycolide (PLGA) andco-polymers thereof.

“Bone” refers to a calcified (mineralized) connective tissue primarilycomprising a composite of deposited calcium and phosphate in the form ofhydroxyapatite, collagen (primarily Type I collagen) and bone cells suchas osteoblasts, osteocytes and osteoclasts, as well as to bone marrowtissue which forms in the interior of true endochondral bone. Bonetissue differs significantly from other tissues, including cartilagetissue. Specifically, bone tissue is vascularized tissue composed ofcells and a biphasic medium comprising a mineralized, inorganiccomponent (primarily hydroxyapatite crystals) and an organic component(primarily of Type I collagen). Glycosaminoglycans constitute less than2% of this organic component and less than 1% of the biphasic mediumitself, or of bone tissue per se. Moreover, relative to cartilagetissue, the collagen present in bone tissue exists in a highly-organizedparallel arrangement. Bony defects, whether from degenerative, traumaticor cancerous etiologies, pose a formidable challenge to thereconstructive surgeon. Particularly difficult is reconstruction orrepair of skeletal parts that comprise part of a multi-tissue complex,such as occurs in mammalian joints.

“Bone formation” means formation of endochondral bone or formation ofintramembranous bone. In humans, bone formation begins during the first6-8 weeks of fetal development. Progenitor stem cells of mesenchymalorigin migrate to predetermined sites, where they either: (a) condense,proliferate, and differentiate into bone-forming cells (osteoblasts), aprocess observed in the skull and referred to as “intramembranous boneformation” or, (b) condense, proliferate and differentiate intocartilage-forming cells (chondroblasts) as intermediates, which aresubsequently replaced with bone-forming cells. More specifically,mesenchymal stem cells differentiate into chondrocytes. The chondrocytesthen become calcified, undergo hypertrophy and are replaced by newlyformed bone made by differentiated osteoblasts, which now are present atthe site. Subsequently, the mineralized bone is extensively remodeled,thereafter becoming occupied by an ossicle filled with functionalbone-marrow elements. This process is observed in long bones andreferred to as “endochondral bone formation.” In postfetal life, bonehas the capacity to repair itself upon injury by mimicking the cellularprocess of embryonic endochondral bone development. That is, mesenchymalprogenitor stem cells from the bone-marrow, periosteum, and muscle canbe induced to migrate to the defect site and begin the cascade of eventsdescribed above. There, they accumulate, proliferate, and differentiateinto cartilage, which is subsequently replaced with newly formed bone.

“Bone morphogenic protein (BMP)” refers to a protein belonging to theBMP family of the TGF-β superfamily of proteins (BMP family) based onDNA and amino acid sequence homology. A protein belongs to the BMPfamily according to this invention when it has at least 50% amino acidsequence identity with at least one known BMP family member within theconserved C-terminal cysteine-rich domain which characterizes the BMPprotein family. Members of the BMP family may have less than 50% DNA oramino acid sequence identity overall.

“Conservative substitutions” are residues that are physically orfunctionally similar to the corresponding reference residues. That is, aconservative substitution and its reference residue have similar size,shape, electric charge, chemical properties including the ability toform covalent or hydrogen bonds, or the like. Preferred conservativesubstitutions are those fulfilling the criteria defined for anaccepted-point mutation in Dayhoff et al., supra. Examples ofconservative substitutions are substitutions within the followinggroups: (a) valine, glycine; (b) glycine, alanine; (c) valine,isoleucine, leucine; (d) aspartic acid, glutamic acid; (e) asparagine,glutamine; (f) serine, threonine; (g) lysine, arginine, methionine; and(h) phenylalanine, tyrosine. The term “conservative variant” or“conservative variation” also includes the use of a substituting aminoacid residue in place of an amino acid residue in a given parent aminoacid sequence, where antibodies specific for the parent sequence arealso specific for, i.e., “cross-react” or “immuno-react” with, theresulting substituted polypeptide sequence.

“Defect” or “defect site” refers to a site requiring bone, joint,cartilage or ligament repair, construction, fusion, regeneration oraugmentation. The site may be an orthopedic structural disruption orabnormality, or a site where bone does not normally grow. The defectfurther can define an osteochondral defect, including a structuraldisruption of both the bone and overlying cartilage. A defect can assumethe configuration of a “void”, which is understood to mean athree-dimensional defect such as, for example, a gap, cavity, hole orother substantial disruption in the structural integrity of a bone orjoint. A defect can be the result of accident, disease, surgicalmanipulation, and/or prosthetic failure. In certain embodiments, thedefect is a void having a volume incapable of endogenous or spontaneousrepair. Such defects in long bone are generally twice the diameter ofthe subject bone and are also called “critical size” defects. Forexample, in a canine ulna defect model, the art recognizes such defectsto be approximately 3-4 cm. Generally, critical size defects areapproximately 1.0 cm, and incapable of spontaneous repair. See, forexample, Schmitz et al., Clinical Orthopaedics and Related Research,205, pp. 299-308 (1986); and Vukicevic et al., in Advances in Molecularand Cell Biology, 6, pp. 207-224 (1993)(JAI Press, Inc.). In rabbit andmonkey segmental defect models, the gap is approximately 1.5 cm and 2.0cm, respectively. In other embodiments, the defect is a non-criticalsize segmental defect. Generally, these are capable of spontaneousrepair. In certain other embodiments, the defect is an osteochondraldefect, such as an osteochondral plug. Such a defect traverses theentirety of the overlying cartilage and enters, at least in part, theunderlying bony structure. In contrast, a chondral or subchondral defecttraverses the overlying cartilage, in part or in whole, respectively,but does not involve the underlying bone. Other defects susceptible torepair using the instant invention include, but are not limited to,non-union fractures; bone cavities; tumor resection; fresh fractures(distracted or undistracted); cranial, maxillofacial and facialabnormalities, for example, in facial skeletal reconstruction,specifically, orbital floor reconstruction, augmentation of the alveolarridge or sinus, periodontal defects and tooth extraction socket;cranioplasty, genioplasty, chin augmentation, palate reconstruction, andother large bony reconstructions; vertebroplasty, interbody fusions inthe cervical, thoracic and lumbar spine and posteriolateral fusions inthe thoracic and lumbar spine; in osteomyelitis for bone regeneration;appendicular fusion, ankle fusion, total hip, knee and joint fusions orarthroplasty; correcting tendon and/or ligamentous tissue defects suchas, for example, the anterior, posterior, lateral and medial ligamentsof the knee, the patella and achilles tendons, and the like as well asthose defects resulting from diseases such as cancer, arthritis,including osteoarthritis, and other bone degenerative disorders such asosteochondritis dessicans.

“Granulating solution” refers to a solution that has a certain degree ofconsistency and cohesiveness, and enhances the formation of granules.

“Morphogenic protein” refers to a protein having morphogenic activity(see below). Preferably a morphogenic protein of this inventioncomprises at least one polypeptide belonging to the BMP protein family.Morphogenic proteins may be capable of inducing progenitor cells toproliferate and/or to initiate differentiation pathways that lead tocartilage, bone, tendon, ligament, neural or other types of tissueformation depending on local environmental cues, and thus morphogenicproteins may behave differently in different surroundings. For example,an osteogenic protein may induce bone tissue at one treatment site andneural tissue at a different treatment site.

“Morphogenic protein stimulatory factor (MPSF)” refers to a factor thatis capable of stimulating the ability of a morphogenic protein to inducetissue formation from a progenitor cell. The MPSF may have a direct orindirect effect on enhancing morphogenic protein inducing activity. Forexample, the MPSF may increase the bioactivity of another MPSF. Agentsthat increase MPSF bioactivity include, for example, those that increasethe synthesis, half-life, reactivity with other biomolecules such asbinding proteins and receptors, or the bioavailability of the MPSF.

“Osteogenic protein (OP)” refers to a morphogenic protein that iscapable of inducing a progenitor cell to form cartilage and/or bone. Thebone may be intramembranous bone or endochondral bone. Most osteogenicproteins are members of the BMP protein family and are thus also BMPs.As described elsewhere herein, the class of proteins is typified byhuman osteogenic protein (hOP-1). Other osteogenic proteins useful inthe practice of the invention include osteogenically active forms ofOP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-9, DPP, Vg1,Vgr, 60A protein, GDF-1, GDF-3, GDF-5, GDF-6, GDF-7, BMP-10, BMP-11,BMP-13, BMP-15, UNIVIN, NODAL, SCREW, ADMP or NEURAL and amino acidsequence variants thereof. In one currently preferred embodiment,osteogenic protein includes any one of: OP-1, OP-2, OP-3, BMP-2, BMP-4,BMP-5, BMP-6, BMP-9, and amino acid sequence variants and homologsthereof, including species homologs thereof. Particularly preferredosteogenic proteins are those comprising an amino acid sequence havingat least 70% homology with the C-terminal 102-106 amino acids, definingthe conserved seven cysteine domain, of human OP-1, BMP-2, and relatedproteins. Certain preferred embodiments of the instant inventioncomprise the osteogenic protein, OP-1. As further described elsewhereherein, the osteogenic proteins suitable for use with applicants'invention can be identified by means of routine experimentation usingthe art-recognized bioassay described by Reddi and Sampath (Sampath etal., Proc. Natl. Acad. Sci., 84, pp. 7109-13, incorporated herein byreference)

Proteins useful in this invention include eukaryotic proteins identifiedas osteogenic proteins (see U.S. Pat. No. 5,011,691, incorporated hereinby reference), such as the OP-1, OP-2, OP-3 and CBMP-2 proteins, as wellas amino acid sequence-related proteins, such as DPP (from Drosophila),Vg1 (from Xenopus), Vgr-1 (from mouse), GDF-1 (from humans, see Lee,PNAS, 88, pp. 4250-4254 (1991)), 60A (from Drosophila, see Wharton etal. PNAS, 88, pp. 9214-9218 (1991)), dorsalin-1 (from chick, see Basleret al. Cell 73, pp. 687-702 (1993) and GenBank accession number L12032)and GDF-5 (from mouse, see Storm et al. Nature, 368, pp. 639-643(1994)). The teachings of the above references are incorporated hereinby reference. BMP-3 is also preferred. Additional useful proteinsinclude biosynthetic morphogenic constructs disclosed in U.S. Pat. No.5,011,691, incorporated herein by reference, e.g., COP-1, COP-3, COP-4,COP-5, COP-7 and COP-16, as well as other proteins known in the art.Still other proteins include osteogenically active forms of BMP-3b (seeTakao, et al. Biochem. Biophys. Res. Comm., 219, pp. 656-662 (1996)).BMP-9 (see WO95/33830), BMP-15 (see WO96/35710), BMP-12 (seeWO95/16035), CDMP-1 (see WO 94/12814), CDMP-2 (see WO94/12814), BMP-10(see WO94/26893), GDF-1 (see WO92/00382), GDF-10 (see WO95/10539), GDF-3(see WO94/15965) and GDF-7 (see WO95/01802). The teachings of the abovereferences are incorporated herein by reference.

“Repair” is intended to mean new bone and/or cartilage formation whichis sufficient to at least partially fill the void or structuraldiscontinuity at the defect. Repair does not, however, mean, orotherwise necessitate, a process of complete healing or a treatmentwhich is 100% effective at restoring a defect to its pre-defectphysiological/structural/mechanical state.

“Synergistic interaction” refers to an interaction in which the combinedeffect of two or more agents is greater than the algebraic sum of theirindividual effects.

Porous β-TCP

This present invention provides a porous β-TCP having a pore size andgranule size appropriate for bone formation, bone regeneration, and bonerepair at a defect site in a human or animal. The porous β-TCP bodydescribed in this invention comprises β-TCP having a multiplicity ofpores. Each pore is a single separate void partitioned by walls and isnot interconnected. The porous β-TCP body of this invention is distinctfrom the cancellous or fenestrate structures that contain capillary voidpaths or interconnections between adjacent pores. The pore diameter sizeof the porous β-TCP of this invention is in the range of 20-500 μm. Inone embodiment, the pore diameter size is in the range of 410-460 um. Ina preferred embodiment, the pore diameter size is 40-190 μm. In anotherembodiment, the pore diameter size is in the range of 20-95 um. In amore preferred embodiment, the pore diameter is in the range of 50-125μm. These pores provide residence spaces for the infiltrating osteolyticcells and osteoblasts when the β-TCP material is embedded in the livingbody. In one embodiment, the pores are spherical and uniformlydistributed. Spherical pores having a diameter in the range of 20-500 μmare appropriate for osteoblast infiltration. Spherical pores alsoprovide the porous body with the necessary mechanical strength duringthe period that new bone is being synthesized, thus preventing the bonefrom fracturing during this period.

Tricalcium phosphate (TCP) has the formula of Ca₃(PO₄)₂, with the Ca/Pratio being about 1.5. TCP powder has an apatite crystal structure. Uponsintering, the apatite structure converts to the rhombic β-TCPstructure. At high temperatures, the metastable, α-TCP structure canalso form. α-TCP is known to have excessive solubility, which does notpermit the rate of resorption to be complementary to the rate ofsubstitution by the hard tissue. In addition, α-TCP is capable ofgenerating harmful inflammatory responses. In a preferred embodiment,the TCP is sintered at high temperatures of 1100-1200° C. Above 1300°C., TCP is converted to the metastable α-TCP. Sintering the TCP reducesits solubility in body fluids, which leads to a corresponding reductionin its chemical activity so that the porous TCP is well tolerated in thebody and acute inflammatory reactions are avoided. Therefore, the porousβ-TCP is preferably sintered. More preferably the β-TCP comprises β-TCPthat is 95-100% pure.

The porous β-TCP material of the present invention may have any shapeand size. In one embodiment, the porous β-TCP is granular and has aparticle size between 0.1 to 2 mm. In a preferred embodiment, theparticle size is 0.5-1.7 mm. In a more preferred embodiment the particlesize is 1.0-1.7 mm. In a most preferred embodiment, the particle size is0.5-1 mm. β-TCP having a granule size of less than 0.1 mm is notappropriate because it will be readily displaced by flowing body fluids.On the other hand, although bone formation is more obvious in largerparticles, β-TCP having a granule size greater than 2 mm is also notappropriate because too many or excessively large gaps will form betweenthe granules, thus preventing the effective coalescence of the β-TCP tothe newly synthesized bone.

The porosity of the β-TCP influences the resorption rate. If theporosity is too high, the strength of the granules will be decreased. Ifthe porosity is too low, the rate of resorption will be slow. The totalporosity is measured using the mercury intrusion parameter method orequivalent methods. In one embodiment, the total porosity is in therange of 5-80%. In another embodiment, the total porosity is in therange of 40-80%. In a more preferred embodiment, the total porosity is65-75%. In a most preferred embodiment, the total porosity is 70%.

The porous β-TCP of this invention may also be combined with one or morebioactive agents. The bioactive agent may be an agent that enhances bonegrowth or a substance that is medically useful or combinations thereof.It is envisioned that the bioactive agent can include but is not limitedto bone morphogenic proteins, growth factors such as EGF, PDGF, IGF,FGF, TGF-α and TGF-β, cytokines, MPSF, hormones, peptides, lipids,trophic agents and therapeutic compositions including antibiotics andchemotherapeutic agents, insulin, chemoattractant, chemotactic factors,enzymes, enzyme inhibitors. It is also envisioned that bioactive agentssuch as vitamins, cytoskeletal agents, cartilage fragments, allografts,autografts, living cells such as chondrocytes, bone marrow cells,mesenchymal stem cells, tissue transplants, immuno-suppressants may beadded to the porous β-TCP.

In one embodiment, the bioactive agent is a bone morphogenic protein. Ina preferred embodiment, the bone morphogenic protein is OP-1 (BMP-7),OP-2, OP-3, COP-1, COP-3, COP-4, COP-5, COP-7, COP-16, BMP-2, BMP-3,BMP-3b, BMP-4, BMP-5, BMP-6, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13,BMP-14, BMP-15, BMP-16, BMP-17, BMP-18, GDF-1, GDF-2, GDF-3, GDF-5,GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, MP121, dorsalin-1,DPP, Vg-1, Vgr-1, 60A protein, NODAL, UNIVIN, SCREW, ADMP, NEURAL, andTGF-β. In a more preferred embodiment, the morphogenic protein is OP-1.

In another embodiment the morphogenic activity of the bone morphogenicprotein is enhanced by the addition of a MPSF. In a preferred embodimentthe MPSF is selected from the group consisting of insulin-like growthfactor I (IGF-I), estradiol, fibroblast growth factor (FGF), growthhormone (GH), growth and differentiation factor (GDF), hydrocortisone(HC), insulin, progesterone, parathyroid hormone (PTH), vitamin D,retinoic acid and IL-6. In a preferred embodiment, the MPSF is selectedfrom IGF-1, IL-6, FGF, PTH. In a more preferred embodiment, the MPSF isIGF-1.

In another embodiment, the bioactive agent is preferably anantimicrobial or antibiotic including but not limited to erythromycin,bacitracin, neomycin, penicillin, polymyxin B, tetracycline, viomycin,chloromycetin and streptomycin, cefazolin, ampicillin, azactam,tobramycin, clindamycin and gentamycin. The concentrations of theantibiotic to be used are well known in the art. Such antibiotics havebeen known and used in connection with bone cement materials. See, forexample, Hoff et al., J. Bone Joint Surg., 63A, pp. 798, (1981); andDueland et al., Clin. Orthop., 169, pp. 264-268, (1982). The teachingsof these two references are incorporated herein by reference.

In another preferred embodiment, the bioactive agent is a repair cell.In a preferred embodiment, the repair cell is a mammalian cell, morepreferably, a human cell of the same type as that of the tissue beingrepaired or reconstructed. Suitable examples of repair cells includebone cells such as bone marrow stem cells, osteocytes, osteoblasts,osteoclasts and bone progenitor cells. In another embodiment, the cellis transfected with a nucleic acid molecule encoding a BMP.

In yet another preferred embodiment, the bioactive agent is a nucleicacid molecule comprising a sequence encoding a BMP, preferably, OP-1(SEQ ID NO: 10). In a preferred embodiment, the nucleic acid molecule isa RNA or DNA molecule. The nucleic acid sequence encoding the BMP may beinserted in recombinant expression vectors. Examples of vectors includebut are not limited to pBR322, pH717, pH731, pH752, pH754 and pW24. SP6vectors may be used for in vitro transcription of RNA. Transcriptionpromoters useful for expressing the BMP include but are not limited tothe SV40 early promoter, the adenovirus promoter (AdMLP), the mousemetallothionein-I promoter (mMT-I), the Rous sarcoma virus (RSV) longterminal repeat (LTR), the mouse mammary tumor virus long terminalrepeat (MMTV-LTR), and the human cytomegalovirus majorintermediate-early promoter (hCMV). The DNA sequences for all of thesepromoters are known in the art and are available commercially. The DNAsequence may also be inserted in the genome of a recombinant virus suchas, for example recombinant adenovirus, adeno-associated virus orretrovirus. The repair cell or bone progenitor cell is then transfectedor infected with the vector or virus and expresses the BMP protein. Thenucleic acid sequence may transiently or stably transfect the repaircell or bone progenitor cell.

In one embodiment, the nucleic acid molecule is directly injected intothe implant site. Preferably, the nucleic acid is trapped in a carrierselected from the group consisting of mannitol, sucrose, lactose,trehalose, liposomes, proteoliposomes that contain viral envelopeproteins and polylysine-glycoprotein complexes. See, e.g., Ledley, J.Pediatrics 110, pp. 1 (1987); Nicolau et al., Proc. Natl. Acad. Sci.U.S.A., 80, pp. 1068 (1983). In another preferred embodiment, thenucleic acid is transfected or infected into target cells such as boneprogenitor cells and repair cells that have been removed from the body.The transfected cell or infected cells are then re-implanted into thebody.

In a most preferred embodiment, the bioactive agent is encapsulated in abiodegradable agent. As the biodegradable agent is slowly resorbed bythe osteoclast cells, the encapsulated bioactive agent is graduallyreleased into the matrix. At the implant site, one may deliver thebioactive agent through a combination of different biodegradable agents,preferably, differing in the rate of resorption, to achieve a multipleboost delivery system. In another preferred embodiment, thebiodegradable agent is multi-layered. Each layer comprises a differentbiodegradable agent, preferably, differing in the rate of resorption.Methods of encapsulating the bioactive agent include but are not limitedto the emulsion-solvent evaporation method (Grandfils et al., Journal ofBiomedical Materials Research, 26, pp. 467-479 (1992)) and the methoddescribed in Herbert et al., Pharmaceutical Research, 15, pp. 357-361(1998). The above references are incorporated herein by reference. Thelatter method is especially suitable for encapsulating proteins. Othermethods are described in U.S. Pat. Nos. 6,110,503, 5,654,008 and5,271,961, which are incorporated herein by reference. In a preferredembodiment, the OP-1 is stabilized by the addition of lactose during theencapsulation process.

The biodegradable agents of this invention may be in bead or microsphereform. The biodegradable agents can be resorbable biocompatible polymersincluding both natural and synthetic polymers. Natural polymers aretypically absorbed by enzymatic degradation in the body, while syntheticresorbable polymers typically degrade by a hydrolytic mechanism. It ispreferred that the particle size of the biodegradable agent is 20-500μm, preferably, 20-140 μm, more preferably 50-140 μm, and mostpreferably 75-140 μm.

In one embodiment, the biodegradable agent is selected from the groupconsisting of ethylenevinylacetate, natural and synthetic collagen,poly(glaxanone), poly(phosphazenes), polyglactin, polyglactic acid,polyaldonic acid, polyacrylic acids, polyalkanoates, polyorthoesters,poly(L-lactide) (PLLA), poly(D,L-lactide) (PDLLA), polyglycolide (PGA),poly(lactide-co-glycolide (PLGA), poly(ζ-caprolactone),poly(trimethylene carbonate), poly(p-dioxanone),poly(ζ-caprolactone-co-glycolide), poly(glycolide-co-trimethylenecarbonate) poly(D,L-lactide-co-trimethylene carbonate), polyarylates,polyhydroxybutyrate (PHB), polyanhydrides, poly(anhydride-co-imide) andco-polymers thereof, polymers of amino acids, propylene-co-fumarates, apolymer of one or more α-hydroxy carboxylic acid monomers, bioactiveglass compositions, admixtures thereof and any derivatives andmodifications thereof. Preferably, the modification changes less than50% of the overall structure of the polymer.

In a preferred embodiment, the biodegradable agent is selected from thegroup consisting of polyorthoesters, poly(L-lactide) (PLLA),poly(D,L-lactide) (PDLLA), polyglycolide (PGA),poly(lactide-co-glycolide (PLGA), poly(ζ-caprolactone),poly(trimethylene carbonate), poly(p-dioxanone),poly(ζ-caprolactone-co-glycolide), poly(glycolide-co-trimethylenecarbonate), poly(D,L-lactide-co-trimethylene carbonate), polyarylatesand co-polymers thereof.

In another more preferred embodiment, the biodegradable agent isselected from the group consisting of poly(glaxanone),poly(phosphazenes), ethylenevinylacetate, polyglactin, polyglactic acid,polyaldonic acid, polyacrylic acids, polyalkanoates, co-polymers thereofand natural and synthetic collagen.

In yet another more preferred embodiment the biodegradable agent isselected from the group consisting of polyhydroxybutyrate (PHB),anhydrides including polyanhydrides, poly(anhydride-co-imide) andco-polymers thereof, polymers of amino acids, propylene-co-fumarates, apolymer of one or more -hydroxy carboxylic acid monomers, (e.g.α-hydroxy acetic acid (glycolic acid) and/or α-hydroxy propionic acid(lactic acid)), bioactive glass compositions. α-hydroxy propionic acidcan be employed in its d- or l-form, or as a racemic mixture.

In a most preferred embodiment the biodegradable agent ispoly(lactide-co-glycolide) (PLGA). Depending upon the desired rate ofrelease of the bioactive agent, the molar ratio of the lactide,glycolide monomers can be adjusted. In a preferred embodiment, themonomer ratio is 50:50. In general, the higher the molecular weight, theslower the biodegradation. Preferably, the molecular weight range of thepolymer is from about 5,000 to 500,000 daltons, more preferably 10,000to 30,000 daltons.

Method of Producing Porous β-TCP

The invention also relates to a method of producing porous β-TCPgranules. The TCP used in preparing the porous β-TCP is preparedaccording to known methods in the art. The TCP is harvested via a spraydryer, preferably to a particle size of less than 10 μm. If the particlesize is too large, it will interfere with the formation of pores.

The fine TCP powder is then mixed with a pore-forming agent thatdecomposes at high temperature into gaseous decomposition productswithout leaving any solid residue. The pore-forming agents of thisinvention may be in bead or resin form. In one embodiment, thepore-forming agents are selected from thermally decomposable materialsuch as naphthalene, prepolymers of polyacrylates, prepolymers ofpolymethacrylates, polymethyl methacrylate, copolymers of methylacrylate and methyl methacrylate and mixtures thereof, polystyrene,polyethylene glycol, crystalline cellulose powder, fibrous cellulose,polyurethanes, polyethylenes, nylon resins and acrylic resins. In a morepreferred embodiment the pore-forming agent is selected from the groupconsisting of polymethyl methacrylate, polystyrene and polyethyleneglycol. It is preferred that the pore-forming agent creates a pore sizediameter of 20-500 μm, more preferably 40-190 μm, and most preferably50-125 μm after sintering.

The proportion and particle size of the pore-forming agent influencesthe porosity and the pore structure. An excessive amount of thepore-forming agent leads to interconnected pores and a decrease indensity of the β-TCP body and hence mechanical strength of the sinteredbody. A deficiency in the amount of the pore-forming agent may result inthe insufficiently developed pore structure. The proportion ofpore-forming agent is preferably 10-50% by weight, more preferably30-40% by weight, most preferably 37.5% by weight.

A granulating solution is then added to the mixture of TCP powder andpore-forming agent to produce a crumbly mass. This improves the sievingprocedure that follows. Depending on the desired viscosity to beachieved and the aqueous properties of the dispersing medium, thecompound used to form the granulating solution may be selected from thegroup consisting of polyvinyl pyrrolidone, starch, gelatin, polyvinylalcohol, polyethylene oxide, hydroxyethyl cellulose, polyvinyl butyraland cellulose acetate butyrate. Preferably, the compound in thegranulating solution is selected from the group consisting of polyvinylpyrrolidone, starch and gelatin.

The crumbly mass is then sieved to select for a range of granule sizes.The size of the granules selected by the sieving process may be in therange of 250-1700 μm, more preferably 1000-1700 μm, most preferably500-1000 μm. The sieved granules are then dried at 90-110° C., morepreferably at 105° C.

The dried granules are then heated to 700-800° C. to remove thepore-forming agent. The temperature is then raised to 1000-1200° C.,more preferably 1150° C., for sintering. The sintered granules undergo aslow cooling procedure to attain pure crystalline β-TCP. In a preferredembodiment the temperature is lowered from 1150° C. to 39° C. in 6hours. After sintering, weight loss and shrinkage takes place in thesample. Pores are formed in the TCP and the pores are surrounded by theskeleton of sintered TCP. The sintered granules are resieved using thesame size sieve as previously used and mixed with a binder as previouslydescribed to form a moldable putty composition.

Alternatively, the porous β-TCP granules may be prepared by mixing theTCP powder with the pore-forming agent. The mixture is blended toachieve homogeneity and pressed into slugs using a press, rotary tabletmachine or chilsonators. The slugs are heated to 700-800° C. to removethe pore-forming agent and sintered at 1000-1100° C., preferably at1150° C. The porous slugs are then fractured into the appropriateparticle size range of 250-1700 μm, more preferably 1000-1700 μm, andmost preferably 500-1000 μm. The porous granules are then mixed with abinder to form a moldable putty composition.

Moldable Putty Composition

The porous β-TCP of this invention may be combined with a biocompatiblebinder to form a moldable putty composition. The moldable putty may bein the form of a paste or a semi-solid having sufficient viscosity. Themoldable putty composition enables the positioning and shaping withinthe voids, defects or other areas in which new bone growth is desired.The cohesiveness of the putty also prevents the problems of particlemigration associated with grafting materials for orthopedic,maxillofacial and dental applications.

The binder according to this invention must be biodegradable,biocompatible and have fluid flow properties. The binders contemplatedas useful herein include, but are not limited to: art-recognizedsuspending agents, viscosity-producing agents, gel-forming agents andemulsifying agents. Other candidates are agents used to suspendingredients for topical, oral or parental administration. Yet othercandidates are agents useful as tablet binders, disintegrants oremulsion stabilizers. Still other candidates are agents used incosmetics, toiletries and food products. Reference manuals such as theUSP XXII-NF XVII (The Nineteen Ninety U.S. Pharmacopeia and the NationalFormulary (1990)) categorize and describe such agents. Preferred bindersinclude resorbable macromolecules from biological or synthetic sourcesincluding sodium alginate, hyaluronic acid, cellulose derivatives suchas alkylcelluloses including methylcellulose, carboxy methylcellulose,carboxy methylcellulose sodium, carboxy methylcellulose calcium or othersalts, hydroxy alkylcelluloses including hydroxypropyl methylcellulose,hydroxybutyl methylcellulose, hydroxyethyl methylcellulose, hydroxyethylcellulose, alkylhydroxyalkyl celluloses including methylhydroxyethylcellulose, collagen, peptides, mucin, chrondroitin sulfate and the like.

Carboxymethylcellulose (CMC) sodium is a preferred binder. CMC iscommercially available from suppliers such as, but not limited to:Hercules Inc., Aqualon® Division, Delaware; FMC Corporation,Pennsylvania; British Celanese, Ltd., United Kingdom; and Henkel KGaA,United Kingdom. Carboxymethylcellulose sodium is the sodium salt of apolycarboxymethyl ether of cellulose with a typical molecular weightranging from 90,000-700,000. Various grades of carboxymethylcellulosesodium are commercially available which have differing viscosities.Viscosities of various grades of carboxymethylcellulose sodium arereported in Handbook of Pharmaceutical Excipients (2nd Edition),American Pharmaceutical Association & Royal Pharmaceutical Society ofGreat Britain. For example, low viscosity 50-200 cP, medium viscosity400-800 cP, high viscosity 1500-3000 cP. A number of grades ofcarboxymethylcellulose sodium are commercially available, the mostfrequently used grade having a degree of substitution (DS) of 0.7. TheDS is defined as the average number of hydroxyl groups substituted peranhydroglucose unit. It is this DS which determines the aqueoussolubility of the polymer. The degree of substitution and the standardviscosity of an aqueous solution of stated concentration is indicated onany carboxymethylcellulose sodium labeling. Low viscosity CMC (Aqualon®Division, Hercules, Inc., Wilmington, Del.) is currently preferred. Thecurrently preferred degrees of substitution range from 0.65-0.90(DS=0.7, Aqualon® Type 7L).

Aside from binders that are flowable at room temperature, binders alsoinclude reagents such as gelatin, that are solubilized in warm or hotaqueous solutions, and are transformed into a non-flowable gel uponcooling. The gelatin composition is formulated so that the compositionis flowable at temperatures above the body temperature of the mammal forimplant, but transitions to relatively non-flowable gel at or slightlyabove such body temperature.

In one embodiment, the binder of this invention is selected from a classof high molecular weight hydrogels including sodium hyaluronate(˜500-3000 kD), chitosan (˜100-300 kD), poloxamer (˜7-18 kD), andglycosaminoglycan (˜2000-3000 kD). In a preferred embodiment, theglycosaminoglycan is N,O-carboxymethylchitosan glucosamine. Hydrogelsare cross-linked hydrophilic polymers in the form of a gel which have athree-dimensional network. Hydrogel matrices can carry a net positive ornet negative charge, or may be neutral. A typical net negative chargedmatrix is alginate. Hydrogels carrying a net positive charge may betypified by extracellular matrix components such as collagen andlaminin. Examples of commercially available extracellular matrixcomponents include Matrigel™ (Dulbecco's modified eagle's medium with 50μg/ml gentamicin) and Vitrogen™ (a sterile solution of purified,pepsin-solubilized bovine dermal collagen dissolved in 0.012 N HCL). Anexample of a net neutral hydrogel is highly crosslinked polyethyleneoxide, or polyvinyalcohol.

In another embodiment, the binder of this invention may also be selectedfrom a class of polymers selected from the group comprising polylacticacid, polyglycolic acid, co-polymers of polylactic acid and polyglycolicacid, polyhydroxybutyric acid, polymalic acid, polyglutamic acid, andpolylactone. In order to have gradual polymer replacement in thematerial by in situ tissue ingrowth over a several-day to several-weekperiod, the molecular weight of the polymer should be compatible withthe required degradation rate of the polymer.

In another preferred embodiment, the binder is polyethylene glycol. Amixture of low- and high-molecular-weight polyethylene glycols canproduce a paste with the proper viscosity. For example, a mixture ofpolyethylene glycols of molecular weight 400-600 daltons and 1500daltons at the proper ratio would be effective.

In yet another embodiment, the binder is selected from a class ofpolysaccharides with an average molecular weight of about 200,000 to5,000,000 daltons consisting of dextran, dextran sulfate,diethylaminoethyl dextran, dextran phosphate or mixtures thereof. Lowermolecular weight polysaccharides have the advantage of a faster dextranabsorption rate, resulting in earlier exposure of the porous β-TCPmaterial. If it is desired that dextrans remain in the site for anextended period, dextrans of relatively high molecular weight may beused. Other preferred polysaccharides include starch, fractionatedstarch, amylopectin, agar, gum arabic, pullullan, agarose, carrageenan,dextrins, fructans, inulin, mannans, xylans, arabinans, glycogens,glucans, xanthan gum, guar gum, locust bean gum, tragacanth gum, karayagum, and derivatives and mixtures thereof.

In another preferred embodiment, the binder is selected from the groupconsisting of mannitol, white petrolatum, mannitol/dextran combinations,mannitol/white petrolatum combinations, sesame oil, fibrin glue andadmixtures thereof. Fibrin glue is currently a preferred binder, whichcomprises a mixture of mammalian fibrinogen and thrombin. Humanfibrinogen is commercially available in products such as, but notlimited to Tissucol® (Immuno AG, Vienna, Austria), Beriplast®(Behringwerke, Marburg, Germany), Biocoll® (Centre de TransfusionSanguine de Lille, Pours, France) and Transglutine® (CNTS FractionationCentre, Strasbourg, France). Fibrin glue may also be made of fibrinogenand thrombin from other mammalian sources, such as, for example, bovineand murine sources.

It is preferred that the binder is selected from the group consisting ofsodium alginate, hyaluronic acid, sodium hyaluronate, gelatin, collagen,peptides, mucin, chrondroitin sulfate, chitosan, poloxamer,glycosaminoglycan, polysaccharide, polyethylene glycol, methylcellulose,carboxy methylcellulose, carboxy methylcellulose sodium, carboxymethylcellulose calcium, hydroxypropyl methylcellulose, hydroxybutylmethylcellulose, hydroxyethyl methylcellulose, hydroxyethylcellulose,methylhydroxyethyl cellulose, hydroxyethyl cellulose, polylactic acid,polyglycolic acid, co-polymers of polylactic acid and polyglycolic acid,polyhydroxybutyric acid, polymalic acid, polyglutamic acid, polylactone,mannitol, white petrolatum, mannitol/dextran combinations,mannitol/white petrolatum combinations, sesame oil, fibrin glue andadmixtures thereof.

More preferably, the binder is selected from the group consisting ofsodium alginate, hyaluronic acid, methylcellulose, carboxymethylcellulose, carboxy methylcellulose sodium, carboxy methylcellulosecalcium, hydroxypropyl methylcellulose, hydroxybutyl methylcellulose,hydroxyethyl methylcellulose, hydroxyethylcellulose, methylhydroxyethylcellulose, hydroxyethyl cellulose and admixtures thereof. Mostpreferably, the binder is selected from the group consisting of sodiumalginate, hyaluronic acid, carboxy methylcellulose, carboxymethylcellulose sodium and carboxy methylcellulose calcium.

The minimum amount of binder is the amount necessary to give easyformability and provide sufficient particle cohesion and shape retentionduring the period of tissue ingrowth. In one embodiment, the weightratio of porous β-TCP to carboxy methylcellulose sodium is in the rangeof 1:0.1 to 1:1.25. In a preferred embodiment, the ratio of porous β-TCPto CMC sodium is 1:0.4.

The invention also relates to a kit for bone implant comprising theporous β-TCP material of the invention and at least one additionalbioactive agent selected from the group consisting of bone morphogenicproteins and antibiotics. The kit comprising the porous β-TCP materialand a bone morphogenic protein may further comprise a morphogenicprotein stimulatory factor. In one embodiment, the kit further comprisesa binder. In another embodiment, the kit comprises the porous β-TCPmaterial of the invention and a binder.

Bone Morphogenic Protein Family

The BMP family, named for its representative bone morphogenic/osteogenicprotein family members, belongs to the TGF-β protein superfamily. Of thereported “BMPs” (BMP-1 to BMP-18), isolated primarily based on sequencehomology, all but BMP-1 remain classified as members of the BMP familyof morphogenic proteins (Ozkaynak et al., EMBO J., 9, pp. 2085-93(1990)).

The BMP family includes other structurally-related members which aremorphogenic proteins, including the drosophila decapentaplegic genecomplex (DPP) products, the Vg1 product of Xenopus laevis and its murinehomolog, Vgr-1 (see, e.g., Massagué, Annu. Rev. Cell Biol., 6, pp.597-641 (1990), incorporated herein by reference).

The C-terminal domains of BMP-3, BMP-5, BMP-6, and OP-1 (BMP-7) areabout 60% identical to that of BMP-2, and the C-terminal domains ofBMP-6 and OP-1 are 87% identical. BMP-6 is likely the human homolog ofthe murine Vgr-1 (Lyons et al., Proc. Natl. Acad. Sci. U.S.A., 86, pp.4554-59 (1989)); the two proteins are 92% identical overall at the aminoacid sequence level (U.S. Pat. No. 5,459,047, incorporated herein byreference). BMP-6 is 58% identical to the Xenopus Vg-1 product.

Biochemical Structural and Functional

Properties of Bone Morphogenic Proteins

The naturally occurring bone morphogens share substantial amino acidsequence homology in their C-terminal regions (domains). Typically, theabove-mentioned naturally occurring osteogenic proteins are translatedas a precursor, having an N-terminal signal peptide sequence typicallyless than about 30 residues, followed by a “pro” domain that is cleavedto yield the mature C-terminal domain of approximately 100-140 aminoacids. The signal peptide is cleaved rapidly upon translation, at acleavage site that can be predicted in a given sequence using the methodof Von Heijne Nucleic Acids Research, 14, pp. 4683-4691 (1986). The prodomain typically is about three times larger than the fully processedmature C-terminal domain.

Another characteristic of the BMP protein family members is theirapparent ability to dimerize. Several bone-derived osteogenic proteins(OPs) and BMPs are found as homo- and heterodimers in their activeforms. The ability of OPs and BMPs to form heterodimers may conferadditional or altered morphogenic inductive capabilities on morphogenicproteins. Heterodimers may exhibit qualitatively or quantitativelydifferent binding affinities than homodimers for OP and BMP receptormolecules. Altered binding affinities may in turn lead to differentialactivation of receptors that mediate different signaling pathways, whichmay ultimately lead to different biological activities or outcomes.Altered binding affinities could also be manifested in a tissue or celltype-specific manner, thereby inducing only particular progenitor celltypes to undergo proliferation and/or differentiation.

In preferred embodiments, the pair of morphogenic polypeptides haveamino acid sequences each comprising a sequence that shares a definedrelationship with an amino acid sequence of a reference morphogen.Herein, preferred osteogenic polypeptides share a defined relationshipwith a sequence present in osteogenically active human OP-1, SEQ IDNO: 1. However, any one or more of the naturally occurring orbiosynthetic sequences disclosed herein similarly could be used as areference sequence. Preferred osteogenic polypeptides share a definedrelationship with at least the C-terminal six cysteine domain of humanOP-1, residues 335-431 of SEQ ID NO: 1. Preferably, osteogenicpolypeptides share a defined relationship with at least the C-terminalseven cysteine domain of human OP-1, residues 330-431 of SEQ ID NO: 1.That is, preferred polypeptides in a dimeric protein with bonemorphogenic activity each comprise a sequence that corresponds to areference sequence or is functionally equivalent thereto.

Functionally equivalent sequences include functionally equivalentarrangements of cysteine residues disposed within the referencesequence, including amino acid insertions or deletions which alter thelinear arrangement of these cysteines, but do not materially impairtheir relationship in the folded structure of the dimeric morphogenprotein, including their ability to form such intra- or inter-chaindisulfide bonds as may be necessary for morphogenic activity.Functionally equivalent sequences further include those wherein one ormore amino acid residues differs from the corresponding residue of areference sequence, e.g., the C-terminal seven cysteine domain (alsoreferred to herein as the conserved seven cysteine skeleton) of humanOP-1, provided that this difference does not destroy bone morphogenicactivity. Accordingly, conservative substitutions of corresponding aminoacids in the reference sequence are preferred. Amino acid residues thatare conservative substitutions for corresponding residues in a referencesequence are those that are physically or functionally similar to thecorresponding reference residues, e.g., that have similar size, shape,electric charge, chemical properties including the ability to formcovalent or hydrogen bonds, or the like. Particularly preferredconservative substitutions are those fulfilling the criteria defined foran accepted point mutation in Dayhoff et al., supra, the teachings ofwhich are incorporated by reference herein.

Conservative substitutions typically include the substitution of oneamino acid for another with similar characteristics, e.g., substitutionswithin the following groups: valine, glycine; glycine, alanine; valine,isoleucine, leucine; aspartic acid, glutamic acid; asparagine,glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine. The term “conservative variation” also includes the use of asubstituted amino acid in place of an unsubstituted parent amino acidprovided that antibodies raised to the substituted polypeptide alsoimmunoreact with the unsubstituted polypeptide.

The osteogenic protein OP-1 has been described (see, e.g., Oppermann etal., U.S. Pat. No. 5,354,557, incorporated herein by reference).Natural-sourced osteogenic protein in its mature, native form is aglycosylated dimer typically having an apparent molecular weight ofabout 30-36 kDa as determined by SDS-PAGE. When reduced, the 30 kDaprotein gives rise to two glycosylated peptide subunits having apparentmolecular weights of about 16 kDa and 18 kDa. In the reduced state, theprotein has no detectable osteogenic activity. The unglycosylatedprotein, which also has osteogenic activity, has an apparent molecularweight of about 27 kDa. When reduced, the 27 kDa protein gives rise totwo unglycosylated polypeptides, having molecular weights of about 14kDa to 16 kDa, capable of inducing endochondral bone formation in amammal. Osteogenic proteins may include forms having varyingglycosylation patterns, varying N-termini, and active truncated ormutated forms of native protein. As described above, particularly usefulsequences include those comprising the C-terminal 96 or 102 amino acidsequences of DPP (from Drosophila), Vg1 (from Xenopus), Vgr-1 (frommouse), the OP-1 and OP-2 proteins, (see U.S. Pat. No. 5,011,691 andOppermann et al., incorporated herein by reference), as well as theproteins referred to as BMP-2, BMP-3, BMP-4 (see WO88/00205, U.S. Pat.No. 5,013,649 and WO91/18098, incorporated herein by reference), BMP-5and BMP-6 (see WO90/11366, PCT/US90/01630, incorporated herein byreference), BMP-8 and BMP-9.

Preferred morphogenic and osteogenic proteins of this invention compriseat least one polypeptide selected from the group consisting of OP-1(BMP-7), OP-2, OP-3, COP-1, COP-3, COP-4, COP-5, COP-7, COP-16, BMP-2,BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-9, BMP-10, BMP-11, BMP-12,BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, BMP-18, GDF-1, GDF-2, GDF-3,GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, MP121,dorsalin-1, DPP, Vg-1, Vgr-1, 60A protein, NODAL, UNIVIN, SCREW, ADMP,NEURAL, TGF-β and amino acid sequence variants and homologs thereof,including species homologs, thereof. Preferably, the morphogenic proteincomprises at least one polypeptide selected from the group consisting ofOP-1 (BMP-7), BMP-2, BMP-4, BMP-5 and BMP-6; more preferably, OP-1(BMP-7) and BMP-2; and most preferably, OP-1 (BMP-7).

Publications disclosing these sequences, as well as their chemical andphysical properties, include: OP-1 and OP-2 (U.S. Pat. No. 5,011,691;U.S. Pat. No. 5,266,683; Ozkaynak et al., EMBO J., 9, pp. 2085-2093(1990); OP-3 (WO94/10203 (PCT US93/10520)), BMP-2, BMP-3, BMP-4,(WO88/00205; Wozney et al. Science, 242, pp. 1528-1534 (1988)), BMP-5and BMP-6, (Celeste et al., PNAS, 87, 9843-9847 (1991)), Vgr-1 (Lyons etal., PNAS, 86, pp. 4554-4558 (1989)); DPP (Padgett et al. Nature, 325,pp. 81-84 (1987)); Vg-1 (Weeks, Cell, 51, pp. 861-867 (1987)); BMP-9(WO95/33830 (PCT/US95/07084); BMP-10 (WO94/26893 (PCT/US94/05290);BMP-11 (WO94/26892 (PCT/US94/05288); BMP-12 (WO95/16035(PCT/US94/14030); BMP-13 (WO95/16035 (PCT/US94/14030); GDF-1 (WO92/00382(PCT/US91/04096) and Lee et al. PNAS, 88, pp. 4250-4254 (1991); GDF-8(WO94/21681 (PCT/US94/03019); GDF-9 (WO94/15966 (PCT/US94/00685); GDF-10(WO95/10539 (PCT/US94/11440); GDF-11 (WO96/01845 (PCT/US95/08543);BMP-15 (WO96/36710 (PCT/US96/06540); MP-121 (WO96/01316(PCT/EP95/02552); GDF-5 (CDMP-1, MP52) (WO94/15949 (PCT/US94/00657) andWO96/14335 (PCT/US94/12814) and WO93/16099 (PCT/EP93/00350)); GDF-6(CDMP-2, BMP13) (WO95/01801 (PCT/US94/07762) and WO96/14335 andWO95/10635 (PCT/US94/14030)); GDF-7 (CDMP-3, BMP12) (WO95/10802(PCT/US94/07799) and WO95/10635 (PCT/US94/14030)) The above publicationsare incorporated herein by reference. In another embodiment, usefulproteins include biologically active biosynthetic constructs, includingnovel biosynthetic morphogenic proteins and chimeric proteins designedusing sequences from two or more known morphogens.

In another embodiment of this invention, a morphogenic protein may beprepared synthetically for use in concert with a MPSF to induce tissueformation. Morphogenic proteins prepared synthetically may be native, ormay be non-native proteins, i.e., those not otherwise found in nature.

Non-native osteogenic proteins have been synthesized using a series ofconsensus DNA sequences (U.S. Pat. No. 5,324,819, incorporated herein byreference). These consensus sequences were designed based on partialamino acid sequence data obtained from natural osteogenic products andon their observed homologies with other genes reported in the literaturehaving a presumed or demonstrated developmental function.

Several of the biosynthetic consensus sequences (called consensusosteogenic proteins or “COPs”.) have been expressed as fusion proteinsin prokaryotes. Purified fusion proteins may be cleaved, refolded,combined with at least one MPSF (optionally in a matrix or device),implanted in an established animal model and shown to have bone- and/orcartilage-inducing activity. The currently preferred syntheticosteogenic proteins comprise two synthetic amino acid sequencesdesignated COP-5 (SEQ. ID NO: 2) and COP-7 (SEQ. ID NO: 3).

Oppermann et al., U.S. Pat. Nos. 5,011,691 and 5,324,819, which areincorporated herein by reference, describe the amino acid sequences ofCOP-5 and COP-7 as shown below: COP5LYVDFS-DVGWDDWIVAPPGYQAFYCHGECPFPLAD COP7LYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD COP5HFNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA COP7HLNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA COP5 ISMLYLDENEKVVLKYNQEMVVEGCGCRCOP7 ISMLYLDENEKVVLKYNQEMVVEGCGCR

In these amino acid sequences, the dashes (-) are used as fillers onlyto line up comparable sequences in related proteins. Differences betweenthe aligned amino acid sequences are highlighted.

The DNA and amino acid sequences of these and other BMP family membersare published and may be used by those of skill in the art to determinewhether a newly identified protein belongs to the BMP family. NewBMP-related gene products are expected by analogy to possess at leastone morphogenic activity and thus classified as a BMP.

In one preferred embodiment of this invention, the morphogenic proteincomprises a pair of subunits disulfide bonded to produce a dimericspecies, wherein at least one of the subunits comprises a recombinantpeptide belonging to the BMP protein family. In another preferredembodiment of this invention, the morphogenic protein comprises a pairof subunits that produce a dimeric species formed through non-covalentinteractions, wherein at least one of the subunits comprises arecombinant peptide belonging to the BMP protein family. Non-covalentinteractions include Van der Waals, hydrogen bond, hydrophobic andelectrostatic interactions. The dimeric species may be a homodimer orheterodimer and is capable of inducing cell proliferation and/or tissueformation.

In certain preferred embodiments, bone morphogenic proteins usefulherein include those in which the amino acid sequences comprise asequence sharing at least 70% amino acid sequence homology or“similarity”, and preferably 80% homology or similarity, with areference morphogenic protein selected from the foregoing naturallyoccurring proteins. Preferably, the reference protein is human OP-1, andthe reference sequence thereof is the C-terminal seven cysteine domainpresent in osteogenically active forms of human OP-1, residues 330-431of SEQ ID NO: 1. In certain embodiments, a polypeptide suspected ofbeing functionally equivalent to a reference morphogen polypeptide isaligned therewith using the method of Needleman, et al., supra,implemented conveniently by computer programs such as the Align program(DNAstar, Inc.). As noted above, internal gaps and amino acid insertionsin the candidate sequence are ignored for purposes of calculating thedefined relationship, conventionally expressed as a level of amino acidsequence homology or identity, between the candidate and referencesequences. “Amino acid sequence homology” is understood herein toinclude both amino acid sequence identity and similarity. Homologoussequences share identical and/or similar amino acid residues, wheresimilar residues are conservation substitutions for, or “allowed pointmutations” of, corresponding amino acid residues in an aligned referencesequence. Thus, a candidate polypeptide sequence that shares 70% aminoacid homology with a reference sequence is one in which any 70% of thealigned residues are either identical to, or are conservativesubstitutions of, the corresponding residues in a reference sequence. Ina currently preferred embodiment, the reference sequence is OP-1. Bonemorphogenic proteins useful herein accordingly include allelic,phylogenetic counterpart and other variants of the preferred referencesequence, whether naturally-occurring or biosynthetically produced(e.g., including “muteins” or “mutant proteins”), as well as novelmembers of the general morphogenic family of proteins, including thoseset forth and identified above. Certain particularly preferredmorphogenic polypeptides share at least 60% amino acid identity with thepreferred reference sequence of human OP-1, still more preferably atleast 65% amino acid identity therewith.

In another embodiment, useful osteogenic proteins include those sharingthe conserved seven cysteine domain and sharing at least 70% amino acidsequence homology (similarity) within the C-terminal active domain, asdefined herein. In still another embodiment, the osteogenic proteins ofthe invention can be defined as osteogenically active proteins havingany one of the generic sequences defined herein, including OPX (SEQ IDNO: 4) and Generic Sequences 7 (SEQ ID NO: 5) and 8 (SEQ ID NO: 6), orGeneric Sequences 9 (SEQ ID NO: 7) and 10 (SEQ ID NO: 8).

The family of bone morphogenic polypeptides useful in the presentinvention, and members thereof, can be defined by a generic amino acidsequence. For example, Generic Sequence 7 (SEQ ID NO: 5) and GenericSequence 8 (SEQ ID NO: 6) are 97 and 102 amino acid sequences,respectively, and accommodate the homologies shared among preferredprotein family members identified to date, including at least OP-1,OP-2, OP-3, CBMP-2A, CBMP-2B, BMP-3, 60A, DPP, Vg1, BMP-5, BMP-6, Vgr-1,and GDF-1. The amino acid sequences for these proteins are describedherein and/or in the art, as summarized above. The generic sequencesinclude both the amino acid identity shared by these sequences in theC-terminal domain, defined by the six and seven cysteine skeletons(Generic Sequences 7 and 8, respectively), as well as alternativeresidues for the variable positions within the sequence. The genericsequences provide an appropriate cysteine skeleton where inter- orintramolecular disulfide bonds can form, and contain certain criticalamino acids likely to influence the tertiary structure of the foldedproteins. In addition, the generic sequences allow for an additionalcysteine at position 36 (Generic Sequence 7) or position 41 (GenericSequence 8), thereby encompassing the morphogenically active sequencesof OP-2 and OP-3. Generic Sequence 7 Leu Xaa Xaa Xaa Phe Xaa Xaa 1 5 XaaGly Trp Xaa Xaa Xaa Xaa Xaa Xaa Pro 10 15 Xaa Xaa Xaa Xaa Ala Xaa TyrCys Xaa Gly 20 25 Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa 30 35 Xaa XaaXaa Asn His Ala Xaa Xaa Xaa Xaa 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 50 55 Xaa Xaa Xaa Cys Cys Xaa Pro Xaa Xaa Xaa 60 65 Xaa Xaa XaaXaa Xaa Leu Xaa Xaa Xaa Xaa 70 75 Xaa Xaa Xaa Val Xaa Leu Xaa Xaa XaaXaa 80 85 Xaa Met Xaa Val Xaa Xaa Cys Xaa Cys Xaa 90 95wherein each Xaa independently is selected from a group of one or morespecified amino acids defined as follows: “res.” means “residue” and Xaaat res.2 (Tyr or Lys); Xaa at res.3=Val or Ile); Xaa at res.4=(Ser, Aspor Glu); Xaa at res.6=(Arg, Gln, Ser, Lys or Ala); Xaa at res.7=(Asp orGlu); Xaa at res.8=(Leu, Val or Ile); Xaa at res. 11=(Gln, Leu, Asp,His, Asn or Ser); Xaa at res.12=(Asp, Arg, Asn or Glu); Xaa atres.13=(Trp or Ser); Xaa at res.14=(Ile or Val); Xaa at res.15=(Ile orVal); Xaa at res.16 (Ala or Ser); Xaa at res.18=(Glu, Gln, Leu, Lys, Proor Arg); Xaa at res.19=(Gly or Ser); Xaa at res.20=(Tyr or Phe); Xaa atres.21=(Ala, Ser, Asp, Met, His, Gln, Leu or Gly); Xaa at res.23=(Tyr,Asn or Phe); Xaa at res.26=(Glu, His, Tyr, Asp, Gln, Ala or Ser); Xaa atres.28=(Glu, Lys, Asp, Gln or Ala); Xaa at res.30=(Ala, Ser, Pro, Gln,Ile or Asn); Xaa at res.31=(Phe, Leu or Tyr); Xaa at res.33=(Leu, Val orMet); Xaa at res.34=(Asn, Asp, Ala, Thr or Pro); Xaa at res.35=(Ser,Asp, Glu, Leu, Ala or Lys); Xaa at res.36=(Tyr, Cys, His, Ser or Ile);Xaa at res.37=(Met, Phe, Gly or Leu); Xaa at res.38=(Asn, Ser or Lys);Xaa at res.39=(Ala, Ser, Gly or Pro); Xaa at res.40=(Thr, Leu or Ser);Xaa at res.44=(Ile, Val or Thr); Xaa at res.45=(Val, Leu, Met or Ile);Xaa at res.46=(Gln or Arg); Xaa at res.47=(Thr, Ala or Ser); Xaa atres.48=(Leu or Ile); Xaa at res.49=(Val or Met); Xaa at res.50=(His, Asnor Arg); Xaa at res.51=(Phe, Leu, Asn, Ser, Ala or Val); Xaa atres.52=(Ile, Met, Asn, Ala, Val, Gly or Leu); Xaa at res.53=(Asn, Lys,Ala, Glu, Gly or Phe); Xaa at res.54=(Pro, Ser or Val); Xaa atres.55=(Glu, Asp, Asn, Gly, Val, Pro or Lys); Xaa at res.56=(Thr, Ala,Val, Lys, Asp, Tyr, Ser, Gly, Ile or His); Xaa at res.57=(Val, Ala orIle); Xaa at res.58=(Pro or Asp); Xaa at res.59=(Lys, Leu or Glu); Xaaat res.60=(Pro, Val or Ala); Xaa at res.63=(Ala or Val); Xaa atres.65=(Thr, Ala or Glu); Xaa at res.66=(Gln, Lys, Arg or Glu); Xaa atres.67=(Leu, Met or Val); Xaa at res.68=(Asn, Ser, Asp or Gly); Xaa atres.69=(Ala, Pro or Ser); Xaa at res.70=(Ile, Thr, Val or Leu); Xaa atres.71=(Ser, Ala or Pro); Xaa at res.72=(Val, Leu, Met or Ile); Xaa atres.74=(Tyr or Phe); Xaa at res.75=(Phe, Tyr, Leu or His); Xaa atres.76=(Asp, Asn or Leu); Xaa at res.77=(Asp, Glu, Asn, Arg or Ser); Xaaat res.78=(Ser, Gln, Asn, Tyr or Asp); Xaa at res.79=(Ser, Asn, Asp, Gluor Lys); Xaa at res.80=(Asn, Thr or Lys); Xaa at res.82=(Ile, Val orAsn); Xaa at res.84=(Lys or Arg); Xaa at res.85=(Lys, Asn, Gln, His, Argor Val); Xaa at res.86=(Tyr, Glu or His); Xaa at res.87=(Arg, Gln, Gluor Pro); Xaa at res.88=(Asn, Glu, Trp or Asp); Xaa at res.90=(Val, Thr,Ala or Ile); Xaa at res.92=(Arg, Lys, Val, Asp, Gln or Glu); Xaa atres.93=(Ala, Gly, Glu or Ser); Xaa at res.95=(Gly or Ala) and Xaa atres.97=(His or Arg).

Generic Sequence 8 (SEQ ID NO: 6) includes all of Generic Sequence 7 andin addition includes the following sequence (SEQ ID NO: 9) at itsN-terminus: SEQ ID NO: 9 Cys Xaa Xaa Xaa Xaa 1 5

Accordingly, beginning with residue 7, each “Xaa” in Generic Sequence 8is a specified amino acid defined as for Generic Sequence 7, with thedistinction that each residue number described for Generic Sequence 7 isshifted by five in Generic Sequence 8. Thus, “Xaa at res.2=(Tyr or Lys)”in Generic Sequence 7 refers to Xaa at res. 7 in Generic Sequence 8. InGeneric Sequence 8, Xaa at res.2=(Lys, Arg, Ala or Gln); Xaa atres.3=(Lys, Arg or Met); Xaa at res.4=(His, Arg or Gln); and Xaa at res.5=(Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr).

In another embodiment, useful osteogenic proteins include those definedby Generic Sequences 9 and 10, defined as follows.

Specifically, Generic Sequences 9 and 10 are composite amino acidsequences of the following proteins: human OP-1, human OP-2, human OP-3,human BMP-2, human BMP-3, human BMP-4, human BMP-5, human BMP-6, humanBMP-8, human BMP-9, human BMP 10, human BMP-11, Drosophila 60A, XenopusVg-1, sea urchin UNIVIN, human CDMP-1 (mouse GDF-5), human CDMP-2 (mouseGDF-6, human BMP-13), human CDMP-3 (mouse GDF-7, human BMP-12), mouseGDF-3, human GDF-1, mouse GDF-1, chicken DORSALIN, dpp, DrosophilaSCREW, mouse NODAL, mouse GDF-8, human GDF-8, mouse GDF-9, mouse GDF-10,human GDF-11, mouse GDF-11, human BMP-15, and rat BMP3b. Like GenericSequence 7, Generic Sequence 9 is a 97 amino acid sequence thataccommodates the C-terminal six cysteine skeleton and, like GenericSequence 8, Generic Sequence 10 is a 102 amino acid sequence whichaccommodates the seven cysteine skeleton. Generic Sequence 9 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Pro Xaa XaaXaa 15 20 Xaa Xaa Xaa Xaa Cys Xaa Gly Xaa Cys Xaa 25 30 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 35 40 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 4550 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 55 60 Xaa Cys Xaa Pro Xaa XaaXaa Xaa Xaa Xaa 65 70 Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa 75 80 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 Xaa Xaa Xaa Cys Xaa Cys Xaa 95wherein each Xaa is independently selected from a group of one or morespecified amino acids defined as follows: “res.” means “residue” and Xaaat res. 1=(Phe, Leu or Glu); Xaa at res. 2=(Tyr, Phe, His, Arg, Thr,Lys, Gln, Val or Glu); Xaa at res. 3=(Val, Ile, Leu or Asp); Xaa at res.4=(Ser, Asp, Glu, Asn or Phe); Xaa at res. 5=(Phe or Glu); Xaa at res.6=(Arg, Gln, Lys, Ser, Glu, Ala or Asn); Xaa at res. 7=(Asp, Glu, Leu,Ala or Gln); Xaa at res. 8=(Leu, Val, Met, Ile or Phe); Xaa at res.9=(Gly, His or Lys); Xaa at res. 10=(Trp or Met); Xaa at res. 11=(Gln,Leu, His, Glu, Asn, Asp, Ser or Gly); Xaa at res. 12=(Asp, Asn, Ser,Lys, Arg, Glu or His); Xaa at res. 13=(Trp or Ser); Xaa at res. 14=(Ileor Val); Xaa at res. 15=(Ile or Val); Xaa at res. 16=(Ala, Ser, Tyr orTrp); Xaa at res. 18=(Glu, Lys, Gln, Met, Pro, Leu, Arg, His or Lys);Xaa at res. 19=(Gly, Glu, Asp, Lys, Ser, Gln, Arg or Phe); Xaa at res.20=(Tyr or Phe); Xaa at res. 21=(Ala, Ser, Gly, Met, Gln, His, Glu, Asp,Leu, Asn, Lys or Thr); Xaa at res. 22=(Ala or Pro); Xaa at res. 23=(Tyr,Phe, Asn, Ala or Arg); Xaa at res. 24=(Tyr, His, Glu, Phe or Arg); Xaaat res. 26=(Glu, Asp, Ala, Ser, Tyr, His, Lys, Arg, Gln or Gly); Xaa atres. 28=(Glu, Asp, Leu, Val, Lys, Gly, Thr, Ala or Gln); Xaa at res.30=(Ala, Ser, Ile, Asn, Pro, Glu, Asp, Phe, Gln or Leu); Xaa at res.31=(Phe, Tyr, Leu, Asn, Gly or Arg); Xaa at res. 32=(Pro, Ser, Ala orVal); Xaa at res. 33=(Leu, Met, Glu, Phe or Val); Xaa at res. 34=(Asn,Asp, Thr, Gly, Ala, Arg, Leu or Pro); Xaa at res. 35=(Ser, Ala, Glu,Asp, Thr, Leu, Lys, Gln or His); Xaa at res. 36=(Tyr, His, Cys, Ile,Arg, Asp, Asn, Lys, Ser, Glu or Gly); Xaa at res. 37=(Met, Leu, Phe,Val, Gly or Tyr); Xaa at res. 38=(Asn, Glu, Thr, Pro, Lys, His, Gly,Met, Val or Arg); Xaa at res. 39=(Ala, Ser, Gly, Pro or Phe); Xaa atres. 40=(Thr, Ser, Leu, Pro, His or Met); Xaa at res. 41=(Asn, Lys, Val,Thr or Gln); Xaa at res. 42=(His, Tyr or Lys); Xaa at res. 43=(Ala, Thr,Leu or Tyr); Xaa at res. 44=(Ile, Thr, Val, Phe, Tyr, Met or Pro); Xaaat res. 45=(Val, Leu, Met, Ile or His); Xaa at res. 46=(Gln, Arg orThr); Xaa at res. 47=(Thr, Ser, Ala, Asn or His); Xaa at res. 48=(Leu,Asn or Ile); Xaa at res. 49=(Val, Met, Leu, Pro or Ile); Xaa at res.50=(His, Asn, Arg, Lys, Tyr or Gln); Xaa at res. 51=(Phe, Leu, Ser, Asn,Met, Ala, Arg, Glu, Gly or Gln); Xaa at res. 52=(Ile, Met, Leu, Val,Lys, Gln, Ala or Tyr); Xaa at res. 53=(Asn, Phe, Lys, Glu, Asp, Ala,Gln, Gly, Leu or Val); Xaa at res. 54=(Pro, Asn, Ser, Val or Asp); Xaaat res. 55=(Glu, Asp, Asn, Lys, Arg, Ser, Gly, Thr, Gln, Pro or His);Xaa at res. 56=(Thr, His, Tyr, Ala, Ile, Lys, Asp, Ser, Gly or Arg); Xaaat res. 57=(Val, Ile, Thr, Ala, Leu or Ser); Xaa at res. 58=(Pro, Gly,Ser, Asp or Ala); Xaa at res. 59=(Lys, Leu, Pro, Ala, Ser, Glu, Arg orGly); Xaa at res. 60=(Pro, Ala, Val, Thr or Ser); Xaa at res. 61=(Cys,Val or Ser); Xaa at res. 63=(Ala, Val or Thr); Xaa at res. 65=(Thr, Ala,Glu, Val, Gly, Asp or Tyr); Xaa at res. 66=(Gln, Lys, Glu, Arg or Val);Xaa at res. 67=(Leu, Met, Thr or Tyr); Xaa at res. 68=(Asn, Ser, Gly,Thr, Asp, Glu, Lys or Val); Xaa at res. 69=(Ala, Pro, Gly or Ser); Xaaat res. 70=(Ile, Thr, Leu or Val); Xaa at res. 71=(Ser, Pro, Ala, Thr,Asn or Gly); Xaa at res. 2=(Val, Ile, Leu or Met); Xaa at res. 74=(Tyr,Phe, Arg, Thr, Tyr or Met); Xaa at res. 75=(Phe, Tyr, His, Leu, Ile,Lys, Gln or Val); Xaa at res. 76=(Asp, Leu, Asn or Glu); Xaa at res.77=(Asp, Ser, Arg, Asn, Glu, Ala, Lys, Gly or Pro); Xaa at res. 78=(Ser,Asn, Asp, Tyr, Ala, Gly, Gln, Met, Glu, Asn or Lys); Xaa at res.79=(Ser, Asn, Glu, Asp, Val, Lys, Gly, Gln or Arg); Xaa at res. 80=(Asn,Lys, Thr, Pro, Val, Ile, Arg, Ser or Gln); Xaa at res. 81=(Val, Ile, Thror Ala); Xaa at res. 82=(Ile, Asn, Val, Leu, Tyr, Asp or Ala); Xaa atres. 83=(Leu, Tyr, Lys or Ile); Xaa at res. 84=(Lys, Arg, Asn, Tyr, Phe,Thr, Glu or Gly); Xaa at res. 85=(Lys, Arg, His, Gln, Asn, Glu or Val);Xaa at res. 86=(Tyr, His, Glu or Ile); Xaa at res. 87=(Arg, Glu, Gln,Pro or Lys); Xaa at res. 88=(Asn, Asp, Ala, Glu, Gly or Lys); Xaa atres. 89=(Met or Ala); Xaa at res. 90=(Val, Ile, Ala, Thr, Ser or Lys);Xaa at res 91=(Val or Ala); Xaa at res. 92=(Arg, Lys, Gln, Asp, Glu,Val, Ala, Ser or Thr); Xaa at res. 93=(Ala, Ser, Glu, Gly, Arg or Thr);Xaa at res. 95=(Gly, Ala or Thr); Xaa at res. 97=(His, Arg, Gly, Leu orSer). Further, after res. 53 in rBMP3b and mGDF-10 there is an Ile;after res. 54 in GDF-1 there is a T; after res. 54 in BMP3 there is a V;after res. 78 in BMP-8 and Dorsalin there is a G; after res. 37 inhGDF-1 there is Pro, Gly, Gly, Pro.

Generic Sequence 10 (SEQ ID NO: 8) includes all of Generic Sequence 9(SEQ ID NO: 7) and in addition includes the following sequence (SEQ IDNO: 9) at its N-terminus: SEQ ID NO: 9 Cys Xaa Xaa Xaa Xaa 1 5

Accordingly, beginning with residue 6, each “Xaa” in Generic Sequence 10is a specified amino acid defined as for Generic Sequence 9, with thedistinction that each residue number described for Generic Sequence 9 isshifted by five in Generic Sequence 10. Thus, “Xaa at res. 1=(Tyr, Phe,His, Arg, Thr, Lys, Gln, Val or Glu)” in Generic Sequence 9 refers toXaa at res. 6 in Generic Sequence 10. In Generic Sequence 10, Xaa atres. 2=(Lys, Arg, Gln, Ser, His, Glu, Ala, or Cys); Xaa at res. 3=(Lys,Arg, Met, Lys, Thr, Leu, Tyr, or Ala); Xaa at res. 4=(His, Gln, Arg,Lys, Thr, Leu, Val, Pro, or Tyr); and Xaa at res. 5=(Gln, Thr, His, Arg,Pro, Ser, Ala, Gln, Asn, Tyr, Lys, Asp, or Leu).

As noted above, certain currently preferred bone morphogenic polypeptidesequences useful in this invention have greater than 60% identity,preferably greater than 65% identity, with the amino acid sequencedefining the preferred reference sequence of hOP-1. These particularlypreferred sequences include allelic and phylogenetic counterpartvariants of the OP-1 and OP-2 proteins, including the Drosophila 60Aprotein. Accordingly, in certain particularly preferred embodiments,useful morphogenic proteins include active proteins comprising pairs ofpolypeptide chains within the generic amino acid sequence hereinreferred to as “OPX” (SEQ ID NO: 4), which defines the seven cysteineskeleton and accommodates the homologies between several identifiedvariants of OP-1 and OP-2. As described therein, each Xaa at a givenposition independently is selected from the residues occurring at thecorresponding position in the C-terminal sequence of mouse or human OP-1or OP-2. Cys Xaa Xaa His Glu Leu Tyr Val Ser Phe Xaa Asp Leu Gly Trp XaaAsp Trp 1 5 10 15 Xaa Ile Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys GluGly Glu Cys Xaa Phe Pro 20 25 30 35 Leu Xaa Ser Xaa Met Asn Ala Thr AsnHis Ala Ile Xaa Gln Xaa Leu Val His Xaa 40 45 50 55 Xaa Xaa Pro Xaa XaaVal Pro Lys Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala 60 65 70 Xaa Ser ValLeu Tyr Xaa Asp Xaa Ser Xaa Asn Val Ile Leu Xaa Lys Xaa Arg 75 80 85 90Asn Met Val Val Xaa Ala Cys Gly Cys His 95 100wherein Xaa at res. 2=(Lys or Arg); Xaa at res. 3=(Lys or Arg); Xaa atres. 11=(Arg or Gln); Xaa at res. 16=(Gln or Leu); Xaa at res. 19=(Ileor Val); Xaa at res. 23=(Glu or Gln); Xaa at res. 26=(Ala or Ser); Xaaat res. 35=(Ala or Ser); Xaa at res. 39=(Asn or Asp); Xaa at res.41=(Tyr or Cys); Xaa at res. 50=(Val or Leu); Xaa at res. 52=(Ser orThr); Xaa at res. 56=(Phe or Leu); Xaa at res. 57=(Ile or Met); Xaa atres. 58=(Asn or Lys); Xaa at res. 60=(Glu, Asp or Asn); Xaa at res.61=(Thr, Ala or Val); Xaa at res. 65=(Pro or Ala); Xaa at res. 71=(Glnor Lys); Xaa at res. 73=(Asn or Ser); Xaa at res. 75=(Ile or Thr); Xaaat res. 80=(Phe or Tyr); Xaa at res. 82=(Asp or Ser); Xaa at res.84=(Ser or Asn); Xaa at res. 89=(Lys or Arg); Xaa at res. 91=(Tyr orHis); and Xaa at res. 97=(Arg or Lys).

In still another preferred embodiment, useful osteogenically activeproteins have polypeptide chains with amino acid sequences comprising asequence encoded by a nucleic acid that hybridizes, under low, medium orhigh stringency hybridization conditions, to DNA or RNA encodingreference morphogen sequences, e.g., C-terminal sequences defining theconserved seven cysteine domains of OP-1, OP-2, BMP-2, BMP-4, BMP-5,BMP-6, 60A, GDF-3, GDF-6, GDF-7 and the like. As used herein, highstringent hybridization conditions are defined as hybridizationaccording to known techniques in 40% formamide, 5×SSPE, 5× Denhardt'sSolution, and 0.1% SDS at 37° C. overnight, and washing in 0.1×SSPE,0.1% SDS at 50° C. Standard stringent conditions are well characterizedin commercially available, standard molecular cloning texts. See, forexample, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. bySambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press:1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985);Oligonucleotide Synthesis (M. J. Gait ed., 1984): Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); and B. Perbal, APractical Guide To Molecular Cloning (1984), the disclosures of whichare incorporated herein by reference.

As noted above, proteins useful in the present invention generally aredimeric proteins comprising a folded pair of the above polypeptides.Such morphogenic proteins are inactive when reduced, but are active asoxidized homodimers and when oxidized in combination with others of thisinvention to produce heterodimers. Thus, members of a folded pair ofmorphogenic polypeptides in a morphogenically active protein can beselected independently from any of the specific polypeptides mentionedabove.

The bone morphogenic proteins useful in the materials and methods ofthis invention include proteins comprising any of the polypeptide chainsdescribed above, whether isolated from naturally-occurring sources, orproduced by recombinant DNA or other synthetic techniques, and includesallelic and phylogenetic counterpart variants of these proteins, as wellas muteins thereof, and various truncated and fusion constructs.Deletion or addition mutants also are envisioned to be active, includingthose which may alter the conserved C-terminal six or seven cysteinedomain, provided that the alteration does not functionally disrupt therelationship of these cysteines in the folded structure. Accordingly,such active forms are considered the equivalent of the specificallydescribed constructs disclosed herein. The proteins may include formshaving varying glycosylation patterns, varying N-termini, a family ofrelated proteins having regions of amino acid sequence homology, andactive truncated or mutated forms of native or biosynthetic proteins,produced by expression of recombinant DNA in host cells.

The bone morphogenic proteins contemplated herein can be expressed fromintact or truncated cDNA or from synthetic DNAs in prokaryotic oreukaryotic host cells, and purified, cleaved, refolded, and dimerized toform morphogenically active compositions. Currently preferred host cellsinclude, without limitation, prokaryotes including E. coli or eukaryotesincluding yeast, or mammalian cells, such as CHO, COS or BSC cells. Oneof ordinary skill in the art will appreciate that other host cells canbe used to advantage. Detailed descriptions of the bone morphogenicproteins useful in the practice of this invention, including how tomake, use and test them for osteogenic activity, are disclosed innumerous publications, including U.S. Pat. Nos. 5,266,683 and 5,011,691,the disclosures of which are incorporated by reference herein, as wellas in any of the publications recited herein, the disclosures of whichare incorporated herein by reference.

Thus, in view of this disclosure and the knowledge available in the art,skilled genetic engineers can isolate genes from cDNA or genomiclibraries of various different biological species, which encodeappropriate amino acid sequences, or construct DNAs fromoligonucleotides, and then can express them in various types of hostcells, including both prokaryotes and eukaryotes, to produce largequantities of active proteins capable of stimulating endochondral bonemorphogenesis in a mammal.

Morphogenic Protein Stimulatory Factors (MPSF)

A morphogenic protein stimulatory factor (MPSF) according to thisinvention is a factor that is capable of stimulating the ability of amorphogenic protein to induce tissue formation from a progenitor cell.The MPSF may have an additive effect on tissue induction by themorphogenic protein. Preferably, the MPSF has a synergistic effect ontissue induction by the morphogenic protein.

The progenitor cell that is induced to proliferate and/or differentiateby the morphogenic protein of this invention is preferably a mammaliancell. Progenitor cells include mammalian chondroblasts, myoblasts,osteoblasts, neuroblasts and vascular tissue precursor cells, allearlier developmental precursors thereof, and all cells that developtherefrom (e.g., chondroblasts, pre-chondroblasts and chondrocytes).However, morphogenic proteins are highly conserved throughout evolution,and non-mammalian progenitor cells are also likely to be stimulated bysame- or cross-species morphogenic proteins and MPSF combinations. It isthus envisioned that when schemes become available for implantingxenogeneic cells into humans without causing adverse immunologicalreactions, non-mammalian progenitor cells stimulated by morphogenicprotein and a MPSF according to the procedures set forth herein will beuseful for tissue regeneration and repair in humans.

One or more MPSFs are selected for use in concert with one or moremorphogenic proteins according to the desired tissue type to be inducedand the site at which the morphogenic protein and MPSF will beadministered. The particular choice of a morphogenic protein(s)/MPSF(s)combination and the relative concentrations at which they are combinedmay be varied systematically to optimize the tissue type induced at aselected treatment site using the procedures described herein.

The preferred morphogenic protein stimulatory factors (MPSFs) of thisinvention are selected from the group consisting of hormones, cytokinesand growth factors. Most preferred MPSFs for inducing bone and/orcartilage formation in concert with an osteogenic protein comprise atleast one compound selected from the group consisting of insulin-likegrowth factor I (IGF-I), estradiol, fibroblast growth factor (FGF),growth hormone (GH), growth and differentiation factor (GDF),hydrocortisone (HC), insulin, progesterone, parathyroid hormone (PTH),vitamin D (1,25-(OH)₂D₃), retinoic acid and an interleukin, particularlyIL-6.

In another preferred embodiment of this invention, the MPSF comprises acompound or an agent that is capable of increasing the bioactivity ofanother MPSF. Agents that increase MPSF bioactivity include, forexample, those that increase the synthesis, half-life, reactivity withother biomolecules such as binding proteins and receptors, or thebioavailability of the MPSF. These agents may comprise hormones, growthfactors, peptides, cytokines, carrier molecules such as proteins orlipids, or other factors that increase the expression or the stabilityof the MPSF.

For example, when the selected MPSF is IGF-I, agents that increase itsbioactivity include GH, PTH, vitamin D, and cAMP inducers, which maythus function as MPSFs according to this invention. In addition, almostall of the IGF-I in circulation and the extracellular space is bound bya group of high affinity binding proteins called IGFBPs which canaugment or inhibit IGF-I bioactivity (see, e.g., Jones and Clemmons,Endocrine Reviews, 16, pp. 3-34 (1995)). Thus IGFBPs and agents whichalter the levels of IGFBPs such that the bioactive IGF-I concentrationis ultimately increased will also function as a MPSF according to thisinvention.

These or other agents that increase IGF-I bioactivity may be used aloneas the primary MPSF, or one or more may be used as additional MPSFs incombination with IGF-I, to stimulate the tissue inductive activity ofthe morphogenic protein. One such preferred combination comprising atleast two MPSFs for cartilage and bone formation is osteogenic proteinOP-1, IGF-I and PTH.

Preferably, the MPSF is present in an amount capable of synergisticallystimulating the tissue inductive activity of the morphogenic protein ina mammal. The relative concentrations of morphogenic protein and MPSFthat will optimally induce tissue formation when administered to amammal may be determined empirically by the skilled practitioner usingthe procedures described herein.

Implant Device

The invention also relates to an implant device for promoting boneformation, regeneration and repair. The implant device comprises theporous β-TCP material of the invention, and optionally at least onebioactive agent.

The implant device comprising the porous β-TCP material serves as atemporary scaffold and substratum for recruitment of migratoryprogenitor cells, and as a base for their subsequent anchoring andproliferation.

In a preferred embodiment, the implant device comprises the porous β-TCPmatrix and a bioactive agent, which is dispersed or absorbed in thematrix. It is envisioned that the bioactive agent can include but is notlimited to bone morphogenic proteins, growth factors such as EGF, PDGF,IGF, FGF, TGF-α and TGF-β, cytokines, MPSF, hormones, peptides, lipids,trophic agents and therapeutic compositions including antibiotics andchemotherapeutic agents, insulin, chemoattractant, chemotactic factors,enzymes, enzyme inhibitors. It is also envisioned that bioactive agentssuch as vitamins, cytoskeletal agents, autograft, allograft, cartilagefragments, living cells such as chondrocytes, bone marrow cells,mesenchymal stem cells, tissue transplants, immuno-suppressants may beadded to the porous β-TCP.

The porous β-TCP matrix provides a sustained delivery or support systemfor the bioactive agent, which is released over time at the implantationsite as the matrix material is slowly absorbed. In a preferredembodiment, the bioactive agent is encapsulated in the biodegradableagent. The resorption of the biodegradable agent and the gradual releaseof the bioactive agent provides a sustained release system. The dosageand rate of delivery of the bioactive agent may be controlled based onthe nature of the porous matrix, the nature of the biodegradable agentand the nature of the binding interaction between the bioactive agentencapsulated in the biodegradable agent, the porous matrix andbiodegradable agent. In a preferred embodiment, the bioactive agent is abone morphogenic protein or a nucleic acid molecule that encodes BMP. Ina most preferred embodiment, the BMP is OP-1.

In a preferred embodiment, the bioactive agent is a BMP. In a morepreferred embodiment, the BMP is OP-1. The porous β-TCP matrix canprotect the BMP and MPSF from non-specific proteolysis, and canaccommodate each step of the cellular responses involved in progenitorcell induction during tissue development.

Studies have shown that the methodology for combining matrix andmorphogenic proteins plays a role in achieving successful tissueinduction. The optimal ratios of morphogenic protein to MPSF for aspecific combination and tissue type may be determined empirically bythose of skill in the art. Greater amounts may be used for largeimplants. The procedures used to formulate BMP and MPSF into the matrixare sensitive to the physical and chemical state of both the proteinsand the matrix.

In the preferred osteogenic device with porous β-TCP, the osteogenicprotein diffuses out of the matrix into the implantation site andpermits influx and efflux of cells. The osteogenic protein induces theprogenitor cells to differentiate and proliferate. Progenitor cells maymigrate into the matrix and differentiated cells can move out of theporous matrix into the implant site. The sequential cellular reactionsin the interface of the bone matrix/osteogenic protein implants include:binding of fibrin and fibronectin to implanted matrix, migration andproliferation of mesenchymal cells, differentiation of the progenitorcells into chondroblasts, cartilage formation, cartilage calcification,vascular invasion, bone formation, remodeling, and bone marrowdifferentiation. The preferred osteogenic device with porous β-TCPmaterial, can be applied to bone formation in various orthopedic,periodontal, and reconstructive procedures.

The implant device may also comprise a binder in an admixture with thebioactive agent and/or porous β-TCP material. The binder is added toform a moldable putty which may be shaped to fit a defect site or totake the form of a new tissue. The moldable putty composition can beheld in place by the surrounding tissue or masticated muscle. It ispreferred to shape the matrix to span a tissue defect and to take thedesired form of the new tissue. In the case of bone repair of anon-union defect, for example, it is desirable to use dimensions thatspan the non-union. Rat studies show that the new bone is formedessentially having the dimensions of the device implanted. Thus, thematerial may be used for subcutaneous or intramuscular implants. In boneformation procedures, the material is slowly absorbed by the body and isreplaced by bone in the shape of or very nearly the shape of theimplant.

Prosthetic Device

It is also contemplated that the porous β-TCP material of the presentinvention may be-used in a prosthetic device. The prosthetic devicecomprises a surface region that can be implanted adjacent to a targettissue of a mammal, and a composition that is disposed on the surfaceregion. The prosthetic devices will be useful for repairing orthopedicdefects, injuries or anomalies in the treated mammal. Preferably, themammal is a human patient. The prosthetic device may be made from amaterial comprising metal, ceramic or polymer composite material.Preferred devices comprise a load-bearing core selected from Co—Cr—Moalloys, titanium alloys and stainless steel. Preferred prostheticdevices are selected from the group consisting of a hip device, a fusioncage and a maxillofacial device.

The composition comprises the porous β-TCP material of the invention,and optionally, one or more agents selected from the group consisting ofa bioactive agent or a binder dispersed in the porous β-TCP. In apreferred embodiment, the bioactive agent is encapsulated in thebiodegradable agent. In a preferred embodiment, the bioactive agent is aBMP or nucleic acid encoding BMP, more preferably, OP-1. Osteogenicprotein-coated prosthetic devices may enhance the bond strength betweenthe prosthesis and existing bone. (Rueger et al., U.S. Pat. No.5,344,654, incorporated herein by reference). The composition may act asa coating for synthetically constructed bone material, such as for anartificial hip, replacement of diseased bone, correction of defects, oranchoring teeth. The composition is disposed on the surface of theimplant in an amount sufficient to promote enhanced tissue growth intothe surface. The amount of the composition sufficient to promoteenhanced tissue growth may be determined empirically by those of skilledin the art using bioassays described in Rueger et al., U.S. Pat. No.5,344,654, incorporated herein by reference. Preferably, animal studiesare performed to optimize the concentration of the compositioncomponents before a similar prosthetic device is used in the humanpatient.

In another preferred embodiment, the composition is applied to theclinical procedure of total joint arthroplasty in hips, knees, elbowsand other joints, wherein a diseased or damaged natural joint isreplaced by a prosthetic joint. For example, in a total hiparthroplasty, an acetabular cup is inserted with the composition in theacetabular socket of the pelvis to replace the natural acetabulum. Thecup is held in place by the composition and secured by fixation screws.Generally, the cavity or socket conforms to the outer surface of theacetabular cup. The composition can also be applied to total jointrevision surgery, to strengthen the bondage between joint prostheticdevices and the bone.

In yet another preferred embodiment, the composition is applied to aclinical procedure called vertebroplasty. The composition is injectedinto the interior of a vertebral body. This method is used in thetreatment of osteoporosis to increase the density of bone.

In a preferred embodiment, the prosthetic device is selected from thegroup consisting of a fusion cage, a dowel and other devices having apocket or chamber, such as an interbody fusion for containing thecomposition of the present invention. Preferably, the interbody fusiondevice is produced from material selected from the group consisting oftitanium, PEEK (poly(etheretherketone)) and allograft. The interbodyfusion in the cervical, thoracic and lumbar spine can be administeredvia an anterior or posterior approach. Alternatively, the composition ofthis invention can be used without an associated interbody device toachieve interbody fusion.

Spinal fusion cages are placed into the intervertebral space left afterthe removal of a damaged spinal disc to eliminate local motion and toparticipate in vertebral to vertebra bony fusion. As described in U.S.Pat. No. 5,015,247, incorporated herein by reference, the fusion cagesare in the form of a cylindrical hollow member having an outsidediameter larger than the space between two adjacent vertebrae to befused. The interior space within the cylindrical hollow implant can befilled with the composition of this invention. The cylindrical implantscan also include a threaded exterior to permit threaded insertion into atapped bore formed in the adjacent vertebrae. Alternatively, some fusionimplants have been designed to be impacted into the intradiscal space.As described in U.S. Pat. No. 6,146,420, incorporated herein byreference, the fusion device includes opposite end pieces with anintegral central element. The central element has a much smallerdiameter so that the fusion device forms an annular pocket around thecentral element. The composition of this invention can be disposedwithin the annular pocket between the opposite end pieces.

In a preferred embodiment, the prosthetic device is used for repair ofosseous and discoligamentous instability. The composition of thisinvention may be applied to the intervertebral area, resulting insuperior fusion and consequently achieving definitive stabilization of atraumatized motor segment via a single dorsal approach. This applicationmay eliminate the need to undergo a second operation for fractures ofthe thoracolumbar spine, which, at present, is often necessary butinvolves additional high risks. Also, this method avoids the problemsassociated with transplantation of autogenous cancellous bone and itsassociated risk of high morbidity might be avoided. See, e.g., Rueger etal., Orthopäde, 27, pp. 72-79 (1998).

In another preferred embodiment, the prosthetic device is amaxillofacial device. Maxillofacial devices are applied externally tocorrect facial defects resulting from cancer surgery, accidents,congenital deformities. In order to restore the masticatorydeficiencies, a patient with marginal bone mass is first treated withthe composition of this invention to pack and build up the surgicalsite. A maxillofacial anchoring and distracting system, as illustratedin U.S. Pat. No. 5,899,940, incorporated herein by reference, can beapplied to increase the existing bone quality. Fixation devices, such asa standard threaded bone screw and simple pin point tack or self-lockingand threaded bone tack screw device (U.S. Pat. No. 5,971,985,incorporated herein by reference), are used for the retention of tissuegrafts and synthetic membranes to the maxillofacial bone graft site.Once the site has healed, a second surgery is performed to insert theappropriate length endosseous dental implant and to restore masticatoryfunction.

The invention also provides a method for promoting in vivo integrationof an implantable prosthetic device of this invention into a targettissue of a mammal comprising the steps of a) providing on a surface ofthe prosthetic device a composition comprising the porous β-TCPmaterial, optionally, at least one bioactive agent or a binder, and b)implanting the device in a mammal at a locus where the target tissue andthe surface of the prosthetic device are maintained at least partiallyin contact for a time sufficient to permit tissue growth between thetarget tissue and the device.

Method of Inducing Bone Formation and Delivery

The invention also provides a method of inducing bone formation in amammal. The mammal is preferably a human patient. The method comprisesthe step of implanting in the defect site of a mammal a compositioncomprising the porous β-TCP of the invention. In a preferred embodiment,the composition may further comprise a binder and/or a bioactive agent.The defect can be an endochondreal defect, an osteochondral defect or asegmental defect. The method can be applied to other defects which arenot limited to, non-union fractures; bone cavities; tumor resection;fresh fractures (distracted or undistracted); cranial, maxillofacial andfacial abnormalities, for example, in facial skeletal reconstruction,specifically, orbital floor reconstruction, augmentation of the alveolarridge or sinus, periodontal defects and tooth extraction socket;cranioplasty, genioplasty, chin augmentation, palate reconstruction, andother large bony reconstructions; vertebroplasty, interbody fusions inthe cervical, thoracic and lumbar spine and posteriolateral fusions inthe thoracic and lumbar spine; in osteomyelitis for bone regeneration;appendicular fusion, ankle fusion, total hip, knee and joint fusions orarthroplasty; correcting tendon and/or ligamentous tissue defects suchas, for example, the anterior, posterior, lateral and medial ligamentsof the knee, the patella and achilles tendons, and the like as well asthose defects resulting from diseases such as cancer, arthritis,including osteoarthritis, and other bone degenerative disorders such asosteochondritis dessicans. The method may be used in bone augmentation,bone prosthesis, hard tissue implant, bone scaffolding, fixation systems(e.g. screws, sutures, suture anchors, staples, surgical tacks, clips,plates and screws).

The invention also provides a method of delivering a bioactive agent ata site requiring bone formation comprising the step of implanting theporous β-TCP and a bioactive agent at the defect site of a mammal. Themethod of delivering the bioactive agent may further include a binder.In a preferred embodiment, the bioactive agent is encapsulated in abiodegradable agent. In a preferred embodiment, the bioactive agentbelongs to the bone morphogenic protein family. In another preferredembodiment, the bioactive agent is a nucleic acid molecule comprising asequence encoding a BMP. Preferably, the nucleic acid is trapped in acarrier. In yet another embodiment, the bioactive agent is a bone cellor a cell transfected with nucleic acid encoding BMP. In anotherpreferred embodiment, the delivery of the bioactive agent is sustainedrelease. The biodegradable agent is preferably a biocompatible andnon-immunogenic polymer, more preferably, PLGA. The bioactive agent ispreferably OP-1. The release rate of the bioactive agent can becontrolled by altering the molecular weight of the PLGA. The degradationof PLGA commences when water penetrates the cement matrix to hydrolyzelong polymer chains into short water soluble fragments. This results ina reduction in the molecular weight of the PLGA without loss in itsphysical properties. Gradually, further erosion of the polymer leads tothe disruption of the polymer, thereby releasing the bioactive agent.For example, in the case of 10 kD to 30 kD PLGA, the rate of release forOP-1 is one to six weeks.

The invention also describes a method of delivering a bioactive agent ata site requiring cartilage formation comprising implanting at the defectsite of a mammal a composition comprising the bioactive agent andbiodegradable agent, wherein the bioactive agent is encapsulated in thebiodegradable agent. Preferably, the bioactive agent is OP-1 and thebiodegradable agent is PLGA.

EXAMPLE 1 Preparation of Tricalcium Phosphate

A slurry of lime (calcium oxide-hydroxide) is prepared and dilutephosphoric acid is added dropwise to the slurry, which is stirredconstantly. The molar proportion of calcium oxide to phosphoric acid is3:2. The product characteristics are evaluated by X-ray diffraction andadjustments are made to the proportions if required. The resultantslurry is harvested by spray drying. If the slurry is harvested byfiltration, the dried cake is crushed to a fine powder of amorphous TCP.The particle size of the amorphous TCP is preferably smaller than 10 μm.

EXAMPLE 2 Preparation of β-TCP Granule

The TCP powder was mixed with polystyrene beads (NUNC A/S-Denmark)(0-160μm beads). The 10% polyvinyl pyrrolidone (PVP) granulating solution wasprepared by adding PVP C-30 (Plasdone C-30, ISP technologies lot # TX60810) in small portions in a beaker or flask of stirring water untilthe solution was clear. About 37 ml of 10% PVP solution was added to theTCP mixture in 5 ml increments to form a crumbly mass. As illustrated inTable 1, mixtures were prepared with different proportions ofpore-forming beads and TCP. TABLE 1 bead composition (w/w) beads (g) TCP(g) 12.5% 12.5 87.5   25% 12.5 37.5 37.5% 18.75 31.25   50% 23.75 23.75

The crumbly mass was passed through <500 μm, 500-1000 μm, or 1000-1700μm sieves under a vibrating motion to produce wet granules having thecorresponding particle size ranges. The sieved material was dried undervacuum at 105° C. for 2-3 hours.

The dried granules then underwent a burn off cycle to vaporize/carbonizethe pore-forming material and were subsequently sintered at 1150° C. Thetemperature was raised from 39° C. to 300° C. over an 18 hour period,held at 300° C. for 1 hour, elevated to 700° C. over an 18 hour period,held at 700° C. for 2 hours, and elevated to 1150° C. over a 6 hourperiod, and held at 1150° C. for 6 hours, and slow cooled to 39° C. overa 6 hour period. After the sintering cycle, the resultant material wasconfirmed by X-ray diffraction to be porous crystalline β-TCP.

The 37.5% w/w, 500-1000 μm sintered granules were resieved and mixedwith the binder, carboxy methylcellulose sodium to form a moldableputty. The putty mixtures were formed with different proportions ofβ-TCP and CMC. All combinations of β-TCP and CMC produced a putty havingappropriate adherence properties, and did not break up in excess water.The cohesiveness of the putty was enhanced as the CMC proportionincreased. The β-TCP/CMC 1:0.4 (w/w) putty showed the bestcharacteristics for handling. The rheological properties of the varioussamples were determined.

EXAMPLE 3 Rat Model Bioassay for Bone Induction

This assay consists of implanting samples in subcutaneous sites inrecipient rats under ether anesthesia. Male Long-Evans rats, aged 28-32days, may be used. A vertical incision (1 cm) is made under sterileconditions in the skin over the thoracic region, and a pocket isprepared by blunt dissection. Approximately 25 mg of the test sample isimplanted deep into the pocket and the incision is closed with ametallic skin clip. The day of implantation is designated as day one ofthe experiment. Implants are removed at varying times thereafter (i.e.12 days, 18 days). The heterotrophic site allows for the study of boneinduction without the possible ambiguities resulting from the use oforthotropic sites.

Bone growth is determined biochemically by calcium content of theimplant. Calcium content, is proportional to the amount of bone formedin the implant. Bone formation therefore is calculated by determiningthe calcium content of the implant in rats and is expressed as “boneforming units,” where one bone forming unit represents the amount ofprotein that is needed for half maximal bone forming activity of theimplant. Bone induction exhibited by intact demineralized rat bonematrix is considered to be the maximal bone differentiation activity forcomparison purposes in this assay.

Cellular Events During Endochondral Bone Formation

Successful implants exhibit a controlled progression through the stagesof protein-induced endochondral bone development, including: (1)transient infiltration by polymorphonuclear leukocytes; (2) mesenchymalcell migration and proliferation; (3) chondrocyte appearance; (4)cartilage matrix formation; (5) cartilage calcification; (6) vascularinvasion, appearance of osteoblasts, and formation of new bone; (7)appearance of osteoclasts, bone remodeling and dissolution of theimplanted matrix; and (8) hematopoietic bone marrow differentiation inthe ossicles. This time course in rats may be accelerated by increasingthe amounts of OP-1 added. It is possible that increasing amounts of oneor more MPSFs may also accelerate this time course. The shape of the newbone conforms to the shape of the implanted matrix.

Histological Evaluation

Histological sectioning and staining is preferred to determine theextent of osteogenesis in the implants. Implants are fixed in BouinsSolution, embedded in paraffin, and cut into 6-8 μm sections. Stainingwith toluidine blue or hemotoxylin/eosin demonstrates clearly theultimate development of endochondral bone. Twelve-day implants areusually sufficient to determine whether the implants containnewly-induced bone.

Biological Markers

Alkaline phosphatase (AP) activity may be used as a marker forosteogenesis. The enzyme activity may be determinedspectrophotometrically after homogenization of the implant. The activitypeaks at 9-10 days in vivo and thereafter slowly declines. Implantsshowing no bone development by histology have little or no alkalinephosphatase activity under these assay conditions. The assay is usefulfor quantification and obtaining an estimate of bone formation quicklyafter the implants are removed from the rat. Alternatively, the amountof bone formation can be determined by measuring the calcium content ofthe implant.

Gene expression patterns that correlate with endochondral bone or othertypes of tissue formation can also be monitored by quantitating mRNAlevels using procedures known to those of skill in the art such asNorthern Blot analysis. Such developmental gene expression markers maybe used to determine progression through tissue differentiation pathwaysafter osteogenic protein/MPSF treatments. These markers includeosteoblastic-related matrix proteins such as procollagen α₂ (I),procollagen α₁ (I), procollagen α₁ (III), osteonectin, osteopontin,biglycan, and alkaline phosphatase for bone regeneration (see e.g., Suvaet al., J. Bone Miner. Res., 8, pp. 379-88 (1993); Benayahu et al., J.Cell. Biochem., 56, pp. 62-73 (1994)).

EXAMPLE 4 Sheep Model Bioassay for Bone Repair

Skeletally mature female sheep were included in the study. Three drilleddefects were created in the area of the proximal metaphysis for both theleft and right tibia of each animal. Defects were 6 mm in diameter andat least 10 mm deep. The defect size was consistent across all testanimals. The defects were created so as to maintain the structure of theinterosseous fibrofatty marrow. This marrow acts as a barrier betweenthe implant materials and prevents interosseous mixing of the matrixmaterials tested. As illustrated in Table 2, β-TCP putty I, II, III, IVand collagen were tested in the defect sites with and without OP-1. OP-1was either directly added to the β-TCP formulations or encapsulated inPLGA. Table 3 represents examples of formulations wherein the OP-1 isencapsulated in PLGA. Of the six defect sites in each animal, one defectsite served as a control, which contained no test material.

A 3 to 4 inch incision was made over the proximal tibial metaphysis. Theskin and underlying muscle were dissected to expose the periosteum. Theperiosteum was incised and maintained intact for surgical closure ifpossible. Three transverse holes were created in the metaphysis. Thefirst and most superior was created approximately 2 cm below thearticular surface of the tibia. The defects were created so as to form aline oriented with the long axis of the bone. Implants were spaced at1.6 cm intervals measured center-to-center.

Materials were harvested at four and eight weeks post-treatment. Animalswere euthanised with pentobarbital 75-100 mg/kg IV. The proximal tibiawere taken and cut to best allow for tissue fixation. Specimens werefixed in 10% neutral buffered Formalin. Specimens were cut, if feasible,so as to capture all implant sites in a single specimen. Followingfixation, specimens were decalcified, embedded in plastic and sectionedin longitudinal orientation using Exackt technique and ground toappropriate section thickness for histologic interpretation.

Radiographic assessment (FIGS. 9-16, 27 and 28) and histologicevaluation (FIGS. 1-8) were made at post-operative, four and eight weekson all implant sites. Anterior posterior radiographs were taken so as tobest image all three defects simultaneously and view the cylindricaldefects from the side. Qualitative histologic descriptions identifiednew bone formation, residual implant material and any evidence ofpathologic response. Images were captured for each specimen and scorespresented for bone formation, acute and chronic inflammation andresidual matrix.

Specimen handling and hemostatic properties were recorded at the time ofimplantation. Materials ranged in form and consistency from a putty orgranular form to a semi-solid cylinder. TABLE 2 Initial pore-former CodeFormulation percentage/Granule size 89A β-TCP Putty I 12.5% (w/w), 0.5-1mm 89B β-TCP Putty II   25% (w/w), 0.5-1 mm 89C β-TCP Putty III 37.5%(w/w), 0.5-1 mm 89F β-TCP Putty IV   25% (w/w), 1-2 mm 48C CollagenBovine type I collagen SOBl Lyophil 1 OP-1 SOP2 Lyophil 2 PlaceboReconstitution Resconstitution medium

TABLE 3 Code Formulation formula- β-TCP, 7% (w/w) PLGA (10 kD) with 0.3%(w/w) tion 4 OP-1 formula- β-TCP, 7% (w/w) PLGA (25-30 kD) with 0.3%tion 5 (w/w) OP-1Formulation Handling

Lyophil 1 and Lyophil 2 (placebo) were reconstituted by adding 2.5 ml ofthe reconstitution medium to one vial of the Lyophil (All componentswere stored frozen at 2 to 8° C. until use), shaking the medium gentlyfor 2 minutes until a homogenous (clear to cloudy) gel was formed. 0.4ml of reconstituted Lyophil gel was added to the porous β-TCP matrixslowly and with care. Utilizing a thin spatula, the porous β-TCP matrixwas mixed with the gel to form a putty-like material.

The PLGA microspheres (particle size 75-150 μm, Alkermes, Inc.)encapsulated with 0.3% (w/w) OP-1 were mixed with the porous β-TCPmatrix.

The putty material was immediately implanted. The implant materials wereplaced through the use of a folded piece of sterile paper. The paper wasfilled with test material and used to pour it into the defect whilecontinuously packing material in the site. The handling properties priorto placement and in the defect site were recorded.

The β-TCP Putty I, II, III, IV formulations were poured as a granulardry powder. Once combined with the vehicle solution, the putties had adry crunchy granular texture. The formulations absorbed all of theLyophil solution. The formulation was implanted with a spatula. Once inthe implant site, the materials became well filled with blood.

The collagen formulation poured as a fluffy powder. Once mixed with avehicle solution, it had a gritty putty texture. The formulation couldbe easily placed with a syringe in the implant site. The implant sitebecame well filled with blood.

Histologic Results

Proximal tibia sections contained three defects. These defects weregross macro-cut so that all three were contained in a single section.Based on gross section observations, clinical assays, and faxitronx-rays of this section, the section was considered representative of thesample. This orientation allowed the evaluation of the periostealreaction overlying the defects and intramedullary response to the testmaterials. Specimens were evaluated from 4 and 8 week explants (FIGS.1-8). All three defects within a single tibial section received eitherthe placebo or OP-1 solution. This segregation of the placebo and OP-1implants facilitated the determination of the active or inactivebiologic nature of the implant material.

Four-Week Evaluation for OP-1 and Placebo Implant Materials

At four weeks, the β-TCP Putty I (89A) was present in all sites (FIG. 3middle site and FIG. 2 distal site). Generally, the matrix was notsignificantly resorbed nor was it undergoing active resorption. Sitestreated with OP-1 resulted in some but not marked new bone formation(FIG. 3 middle site). Placebo treated sites had bone formation at thelevel of the cortex (FIG. 2 distal site).

The β-TCP Putty II (89B) was present in all sites at 4 weeks insignificant amounts (FIG. 3 distal site and FIG. 1 proximal site). Therewas no significant evidence of matrix resorption. OP-1 treated sitesresulted in small amounts of new bone formation predominately at thecortical and periosteal level (FIG. 3 distal site). Of the four β-TCPputty formulations tested, β-TCP putty II resulted in more inflammationthan the other three formulations. Foreign body giant cells (FBGC) werereported in conjunction with this inflammation.

β-TCP Putty III (89C) was present in significant amounts in all sixsites treated at 4 weeks (FIG. 1 middle site and FIG. 4 proximal site).OP-1 treatment did not noticeably alter residual matrix volumes. Boneformation at the cortical level was apparent in OP-1 treated specimens(FIG. 4 proximal site) and less common in placebo treated sites (FIG. 1middle site). Little or no inflammation was observed in response to theβ-TCP matrix independent of OP-1 treatment.

β-TCP Putty IV (89F) was present in significant amounts in all six sitestreated at 4 weeks (FIG. 1 distal site and FIG. 4 middle site). OP-1treatment had no apparent effect on residual matrix volume. OP-1 treatedsites resulted in greater bone formation throughout the matrix withcortical and periosteal responses apparent (FIG. 4 middle site). Littleor no inflammation was observed in response to the β-TCP matrixindependent of OP-1 treatment.

Eight-Week Evaluation for OP-1 and Placebo Treated Implant Materials

The β-TCP Putty I (89A) was present in all sites at 8 weeks (FIG. 7proximal site and FIG. 6 distal site). The OP-1 treated implantsgenerally showed evidence of a strong bone inductive response (FIG. 7proximal site). In two OP-1 treated sites, the β-TCP matrix appeared tohave significantly degraded. Sites treated with OP-1 resulted in markednew bone formation at the cortical level with modest bone infiltrationinto the matrix within the medullary space. Placebo treated sitesresulted in less bone formation at the level of the cortex (FIG. 6distal site).

The β-TCP Putty II (89B) was present in all sites at 8 weeks insignificant amounts (FIG. 5 proximal site and FIG. 7 middle site). Therewas no significant evidence of matrix resorption. OP-1 treated sitesresulted in small amounts of new bone formation predominately at thecortical and periosteal level and closure at the defect site (FIG. 7middle site). Placebo treated materials resulted in less bone formationat the cortical level and calcium particles blocking closure of thecortical defect (FIG. 5 proximal site). The inflammation notedpreviously in response to this material was not evident.

β-TCP Putty III (89C) was present in significant amounts in all sixsites treated at 8 weeks (FIG. 5 middle site and FIG. 7 distal site).OP-1 treatment did not noticeably alter residual matrix volumes. Boneformation at the cortical level and a marked periosteal response wasapparent in OP-1 treated specimens (FIG. 7 distal site). Little or noinflammation was observed in response to the β-TCP matrix independent ofOP-1 treatment.

β-TCP Putty IV (89F) was present in significant amounts in all six sitestreated at 8 weeks (FIG. 5 distal site and FIG. 8 proximal site). A fewsites had less residual matrix than others. Generally, OP-1 treatmenthad no apparent effect on residual matrix volume. OP-1 treated sitesresulted in greater bone formation throughout the matrix with anapparent cortical and periosteal response (FIG. 8 proximal site). Littleor no inflammation was observed in response to the β-TCP matrixindependent of OP-1 treatment.

Conclusion of the Above Results

Compared to the collagen material which demonstrated acute and chronicinflammation coupled with an FBGC response, the four porous β-TCPformulations resulted in little or no inflammation at four and eightweeks. OP-1 treatment in the porous β-TCP materials consistentlyexhibited marked bone formation at the cortical level and a reactiveperiosteal response that often resulted in cortical defect closure.Although the large granular (1-2 mm) β-TCP putty IV formulation appearedto allow bone ingrowth deeper in the matrix, there was greaterinter-granular spacing compared to that observed in small granular β-TCPputties.

Paraffin Histology Study

Tissues from the sheep model bioassay were evaluated using paraffinsections and hematoxylin and eosin stain to evaluate the effect ofparticle size and porosity of the implant material on bone formation inand around particles.

Tibial specimens were sectioned so as to isolate implant sites in theproximal, middle and distal sites from four animals (138, 299, 297, and295). These explants were decalcified, embedded in paraffin, sectionedand stained with hematoxylin and eosin.

Sections were viewed using light microscopy and interpreted for theeffect of particle size and porosity. For specimens stratified in boneformation, the response from the cortical level was robust and deep, andthe response was modest in the medullary compartment. Due to thisstratification, the level extending from the endosteal cortex to a level2-3 mm deep was evaluated.

Each of the four ceramic formulations were evaluated for bone formationin the pores and bone bridging across the particles. Bone formation inpores was assessed by counting pores that were completely isolatedwithin a particle from the adjacent stroma. Pores that were obvious andgenerally round were counted. As pores were counted, a ratio was formedof those that had bone over those that did not. This is noted as thepore-fill ratio.

Pore counting was performed by scanning the field. In materials with fewpores, the majority were counted as the field was scanned (FIG. 25). Inmaterials with many pores, regions were counted and a new region wasviewed and then counted (FIG. 26). The average of the regions or totalcount were presented in the ratio.

Bone bridging between particles was scored 0 to 2. A zero score wasgiven to particles when the bone did not bridge to adjacent particles. Ascore of 1 was given when a couple to a few particles consistentlyshowed bridging. A score of 2 was given when many of the particles werejoined by vital bone trabeculae.

Tables 4 and 5 illustrate the pore-fill ratios and bone bridging scoresfor placebo and OP-1 at four weeks (FIGS. 17-20). Tables 6 and 7illustrate the pore-fill ratios and bone bridging scores for placebo andOP-1 at eight weeks (FIGS. 21-24). Bone bridging was more pronounced forβ-TCP putties made from 37.5% (w/w) pore-forming agent and having thesmaller 0.5-1 mm granule size(Tables 4-7). The pore-fill ratio wasgenerally equivalent for the β-TCP putty made from 25% and 37.5% (w/w)pore-forming agents. The β-TCP made from 12.5% (w/w) pore-forming agenthad a lower pore-fill ratio (Tables 4-7). The pore-fill ratio wasconsistently higher in the 89F formulation due to the larger size of theparticle (1-2 mm) with more pores per particle. Compared to the smallparticles (0.5-1 mm), there was less bone bridging in the largerparticles due to the fact that more bone was required to bridge largeparticles. TABLE 4 Initial Pore Treat- Particle Pore- Duration Fill BoneSection ment Size former % (wks) Ratio Bridging 297R-D 89A .5-1 mm 12.54  2/10 0 297L-P 89B .5-1 mm 25 4  6/10 0 297L-M 89C .5-1 mm 37.5 4 6/70 297L-D 89F  1-2 mm 25 4 10/10 0Note:Section 297R-D is from the right side (R), distal (D) site of animal297.Section 297L-P is from the left side(L), proximal site (P) of animal297.Section 297L-M is from the left side(L), middle site (M) of animal 297.Section 297L-D is from the left side(L), distal site (D) of animal 297.

TABLE 5 Initial Pore Treat- Particle Pore- Duration Fill Bone Sectionment Size former % (wks) Ratio Bridging 295L-M 89A .5-1 mm 12.5 4  6/112 295L-D 89B .5-1 mm 25 4  8/11 1 295R-P 89C .5-1 mm 37.5 4 6/8 2 295R-M89F  1-2 mm 25 4 10/10 2Note:Section 295L-M is from the left side (L), middle (M) site of animal 295.Section 295L-D is from the left side(L), distal site (D) of animal 295.Section 295R-P is from the right side(R), proximal site (P) of animal295.Section 295R-M is from the right side(R), middle site (M) of animal 295.

TABLE 6 Initial Pore Treat- Particle Pore- Duration Fill Bone Sectionment Size former % (wks) Ratio Bridging 299R-D 89A .5-1 mm 12.5 8  4/141 299L-P 89B .5-1 mm 25 8  9/10 2 299L-M 89C .5-1 mm 37.5 8 18/20 2299L-D 89F  1-2 mm 25 8  9/10 1Note:Section 299R-D is from the right side (R), distal (D) site of animal299.Section 299L-P is from the left side(L), proximal site (P) of animal299.Section 299L-M is from the left side(L), middle site (M) of animal 299.Section 299L-D is from the left side(L), distal site (D) of animal 299.

TABLE 7 Initial Pore Treat- Particle Pore- Duration Fill Bone Sectionment Size former % (wks) Ratio Bridging 138L-P 89A .5-1 mm 12.5 8 10/201 138L-M 89B .5-1 mm 25 8 8/9 2 138L-D 89C .5-1 mm 37.5 8 10/12 2 138R-P89F  1-2 mm 25 8  9/10 1Note:Section 138L-P is from the left side (L), proximal (P) site of animal138.Section 138L-M is from the left side(L), middle site (M) of animal 138.Section 138L-D is from the left side(L), distal site (D) of animal 138.Section 138R-P is from the right side(R), proximal site (P) of animal138.Conclusion of Paraffin Histology Study

For β-TCP formulations, bone formation in pores became more apparent asthe porosity increased. Bone formation in pores was less frequent in thematerial made from 12.5% pore-former compared to the material made from37.5% pore-former. Although bone formation was more obvious in largerparticles (1-2 mm), less bone bridging was observed in these largeparticles.

The collagen formulations resulted in no bone formation and a markedpathologic response. Moreover, these formulations resulted in a markedFBGCR and chronic fibroinflammatory response.

EXAMPLE 5 Feline Model Bioassay for Bone Repair

A femoral osteotomy defect is surgically prepared. Without furtherintervention, the simulated fracture defect would consistently progressto non-union. The effects of osteogenic compositions and devicesimplanted into the created bone defects are evaluated by the followingstudy protocol.

Briefly, the procedure is as follows: Sixteen adult cats each weighingless than 10 lbs. undergo unilateral preparation of a 1 cm bone defectin the right femur through a lateral surgical approach. In otherexperiments, a 2 cm bone defect may be created. The femur is immediatelyinternally fixed by lateral placement of an 8-hole plate to preserve theexact dimensions of the defect. Four different types of materials may beimplanted in the surgically created cat femoral defects: group I is anegative control group with no test material; group II is implanted withbiologically active porous β-TCP; group III is implanted with porousβ-TCP and an osteogenic protein; and group IV is implanted with porousβ-TCP, an osteogenic protein and MPSF.

All animals are allowed to ambulate ad libitum within their cagespost-operatively. All cats are injected with tetracycline (25 mg/kgsubcutaneously (SQ) each week for four weeks) for bone labeling.

In vivo radiomorphometric studies are carried out immediately at 4, 8,12 and 16 weeks post-operative by taking a standardized X-ray of thelightly-anesthetized animal positioned in a cushioned X-ray jig designedto consistently produce a true anterio-posterior view of the femur andthe osteotomy site. All X-rays are taken in exactly the same fashion andin exactly the same position on each animal. Bone repair is calculatedas a function of mineralization by means of random point analysis. Afinal specimen radiographic study of the excised bone is taken in twoplanes after sacrifice.

Excised test and normal femurs may be immediately studied by bonedensitometry, or wrapped in two layers of saline-soaked towels, placedinto sealed plastic bags, and stored at −20° C. until further study.Bone repair strength, load-to-failure, and work-to-failure are tested byloading to failure on a specially designed steel 4-point bending jigattached to an Instron testing machine to quantitate bone strength,stiffness, energy absorbed and deformation to failure. The study of testfemurs and normal femurs yields the bone strength (load) in pounds andwork-to-failure in joules. Normal femurs exhibit a strength of 96(+/−12) pounds. Osteogenic device-implanted femur strength should becorrected for surface area at the site of fracture (due to the“hourglass” shape of the bone defect repair). With this correction, theresult should correlate closely with normal bone strength.

Following biomechanical testing, the bones are immediately sliced intotwo longitudinal sections at the defect site, weighed, and the volumemeasured. One-half is fixed for standard calcified bonehistomorphometrics with fluorescent stain incorporation evaluation, andone-half is fixed for decalcified hemotoxylin/eosin stain histologypreparation.

Selected specimens from the bone repair site are homogenized in cold0.15 M NaCl, 3 mM NaHCO₃, pH 9.0 by a Spex freezer mill. The alkalinephosphatase activity of the supernatant and total calcium content of theacid soluble fraction of sediment are then determined.

EXAMPLE 6 Rabbit Model Bioassay for Bone Repair

This assay is described in detail in Oppermann et al., U.S. Pat. No.5,354,557; see also Cook et al., J. of Bone and Joint Surgery, 76-A, pp.827-38 (1994), which are incorporated herein by reference). Ulnarnon-union defects of 1.5 cm are created in mature (less than 10 lbs) NewZealand White rabbits with epiphyseal closure documented by X-ray. Theexperiment may include implantation of devices into at least eightrabbits per group as follows: group I negative control implants withouttest material; group II implants with porous β-TCP; group III implantswith porous β-TCP and an osteogenic protein; group IV implants withporous β-TCP, osteogenic protein and MPSF combinations. Ulnae defectsare followed for the full course of the eight week study in each groupof rabbits.

In another experiment, the marrow cavity of the 1.5 cm ulnar defect ispacked with activated osteogenic protein in porous β-TCP in the presenceor absence of a MPSF. The bones are allografted in an intercalaryfashion. Negative control ulnae are not healed by eight weeks and revealthe classic “ivory” appearance. In distinct contrast, the osteogenicprotein/MPSF-treated implants “disappear” radiographically by four weekswith the start of remineralization by six to eight weeks. Theseallografts heal at each end with mild proliferative bone formation byeight weeks. This type of device serves to accelerate allograft repair.

Implants treated with osteogenic protein in the presence of a MPSF mayshow accelerated repair, or may function at the same rate using lowerconcentrations of the osteogenic protein. As was described above, therabbit model may also be used to test the efficacy of and to optimizeconditions under which a particular osteogenic protein/MPSF combinationcan induce local bone formation.

EXAMPLE 7 Dog Ulnar Defect Bioassay for Bone Repair

This assay is performed essentially as described in Cook et al.,Clinical Orthopaedics and Related Research, 301, pp. 302-112 (1994),which is incorporated herein by reference). Briefly, an ulnar segmentaldefect model is used to evaluate bone healing in 35-45 kg adult maledogs. Experimental composites comprising 500 mg of porous β-TCP arereconstituted with varying amounts of OP-1 in the absence or presence ofincreasing concentrations of one or more putative MPSFs. Any osteogenicprotein may be used in place of OP-1 in this assay. Implantations atdefect sites are performed with one carrier control and with theexperimental series of OP-1 and OP-1/MPSF combinations being tested.Mechanical testing is performed on ulnae of animals receiving compositesat 12 weeks after implantation. Radiographs of the forelimbs areobtained weekly until the animals are sacrificed at either 12 or 16postoperative weeks. Histological sections are analyzed from the defectsite and from adjacent normal bone.

The presence of one or more MPSFs may increase the rate of bone repairin dog. The presence of one or more MPSFs may also permit the use ofreduced concentrations of osteogenic protein per composite to achievesimilar or the same results.

EXAMPLE 8 Monkey Ulnar and Tibial Defect Bioassay for Bone Repair

This bone healing assay in African green monkeys is performedessentially as described in Cook et al., J. Bone and Joint Surgery, 77A,pp. 734-50 (1995), which is incorporated herein by reference. Briefly, a2.0 cm osteoperiosteal defect is created in the middle of the ulnarshaft and filled with an implant comprising porous β-TCP matricescontaining OP-1 in the absence or presence of increasing concentrationsof one or more putative MPSFs. Experimental composites comprising porousβ-TCP matrices reconstituted with varying amounts of OP-1 in the absenceor presence of increasing concentrations of one or more putative MPSFsare used to fill 2.0 cm osteoperiosteal defects created in the diaphysisof the tibia. Any osteogenic protein may be used in place of OP-1 inthis assay. Implantations at defect sites are performed with one carriercontrol and with the experimental series of OP-1 and OP-1/MPSFcombinations being tested. Mechanical testing is performed on ulnae andtibia of animals receiving composites. Radiographs and histologicalsections are analyzed from the defect sites and from adjacent normalbone as described in Cook et al.

The presence of one or more MPSFs can increase the rate of bone repairin the monkey. The presence of one or more MPSFs may also permit the useof reduced concentrations of osteogenic protein per composite to achievesimilar or the same results.

EXAMPLE 9 Goat Model Fracture Healing Bioassay

This fracture healing assay in sheep is performed essentially asdescribed in Blokhius et al., Biomaterials, 22, pp. 725-730 (2001),which is incorporated herein by reference. A closed midshaft fracture iscreated in the left tibia of adult female goats with a custom-made threepoint bending device. The fractures are stabilized with an externalfixator, which is placed at the lateral side of the tibia. Threedifferent types of materials are implanted in the goat defects viainjection: group I is a negative control group with no test material;group II is implanted with the biologically active porous β-TCP; groupIII is implanted with porous β-TCP and an osteogenic protein; and groupIV is implanted with porous β-TCP and an osteogenic protein encapsulatedin PLGA. The test material is placed in the fractured gap. Mechanicaltesting (four-point non-destructive bending test) is performed on theanimals receiving composites at two weeks and four weeks. After themechanical testing, anterior, posterior, lateral, and medial slices ofthe fracture gap are sawn to perform radiographs and histologicalsections.

EXAMPLE 10 Fusion Assay of an Unstable Motor Segment of the Sheep LumbarSpine

This assay investigates the healing of osseous and discoligamentousinstability. A motor segment of the spine is a functional unitconsisting of two vertebral bodies lying one above the other, and anintervertebral disc.

A trial group consists of 12 sheep. Two control groups of 12 sheep eachare used. The surgical area at the inferior lumbar spine is preparedafter introduction of general anesthesia and placing the animals inprone position. A skin incision of about 12 cm in length above thespinous processes of the inferior lumbar spine is made. Aftertranssection of the subcutis and fascia, the back muscles are moved tothe side.

Intubation anesthesia is applied by intramuscular injection of 1.5 mlxylazine (Rompun®). Further dosage can be administered as needed. Thesedation requires placement of an intravenous indwelling catheter afterpuncturing an ear vein. The anesthesia is introduced through thecatheter by providing 3-5 mg of thiopental (Trapanal®) per killogram ofbody weight. After endotracheal intubation, the animals are ventilatedusing oxygen (30%), nitrous oxide (laughing gas) and isoflurane(Isofluran®). During the entire surgery, the analgesic fentanyldihydrogen citrate (Fentanyl®) having a dosage 0.2-0.4 mg, isadministered. At the same time, relaxation is achieved by administrationof atracurium besilate (Atracurium®) at a dosage of 0.5 mg/kg of bodyweight.

After complete exposure of the pedicles of lumbar vertebral bodies L4 toL6, a bilateral instrumentation of the pedicles L4 and L6 takes place.This is performed by using pedicle screws of 5 mm or 6 mm in diameter,depending on the diameter found in the pedicles. Subsequently, abilateral transpedicular removal of the disc of the cranial motorsegment L4/L5 is performed over the pedicle of L5 under pediculoscopiccontrol. The endplates of the affected vertebral bodies aredecorticated.

Inter- and intracorporal application of test samples occurs via atranspedicular cannula in all 12 sheep of the trial group. Test samplesinclude porous β-TCP, osteogenic protein or osteogenic proteinencapsulated in PLGA in varying concentrations. In the first controlgroup that consists of 12 sheep, only the porous β-TCP is applied. Inthe second control group, autologous spongiosa is administered insteadof the composition of this invention.

Finally, the internal fixator is installed completely. The type of theinternal fixator as well as the necessary instrumentation and surgicalprocedure is standardized and well known to the skilled practitioner.Drains are placed and the wound is closed using absorbable suture forfascia and subcutis as well as skin staples.

During the entire surgical procedure, an x-ray image amplifier isavailable for intraoperative fluoroscopy. This facilitates exactorientation during the execution of the above steps.

Harvesting of the 12 sheep administered with autologous spongiosa iscarried out under anesthesia as follows: the left iliac crest skin andfascia is cut by making a longitudinal incision about 8 cm long. Thegluteal muscles are moved subperiostally and the cancellous bone graftis harvested from the iliac crest after an osteotomy. Bleeding controland placement of a Drain is performed upon closure of the wound inlayers. The harvesting procedure is standard and known to an ordinaryperson skilled in the art.

Clinical Observations

Daily neurologic examinations are performed to evaluate the gait of theanimals as well as neurological deficits that may occur postoperatively.Operative wounds are closely examined each day. Body weights aremeasured preoperatively and at the time of euthanasia.

Radiographic Analysis

Before evaluation, the complete lumbar spine is freshly dissected, andthe internal fixator is carefully removed. Anteroposterior and plainlateral radiographs of the operated spinal segments are obtained underconsistent conditions of milliamperes, kilovolts, and seconds at 0 and 8weeks to assist in fusion evaluation. The status of the fusion areevaluated with use of the grading system documented by Lenke et al., J.Spinal Disord, 5, pp. 433-442 (1992), incorporated herein by reference.With this system, A indicates a big, solid trabeculated bilateral fusionmass (definitely solid); B, a big, solid unilateral fusion mass with asmall contralateral fusion mass (possibly solid); C, a small, thinbilateral fusion mass with an apparent crack (probably not solid); andD, bilateral resorption of the graft or fusion mass with an obviousbilateral pseudarthrosis (definitely not solid).

Additionally, computerized tomography scans are performed to assess thefusion mass in cross sections and in saggital-plane reconstructions. Foreach fusion mass, approximately forty sequential computerized tomographyscans are made with use of two-millimeter slice intervals and subsequentreconstruction in the saggital plane under consistent magnification andradiographic conditions.

Biomechanical Testing

Four specimens of each group are evaluated biomechanically. Afterradiographic analysis, all muscles are carefully removed whilemaintaining the ligamentous and bony structures. The spines are frozenat −20° C. For each of these specimens, the upper half of the uppervertebra and the lower half of the lower vertebra of the motion segmentL4/L5 are embedded in polymethylmethacrylate (Technovit 3040; HeraeusKulzer GmbH, Wehrheim/Ts, Germany). Each specimen is then fixed andtested without preload in a spine tester in a non-destructive testingmode. Alternating sequences of flexion/extension, axial right/leftrotation, and right/left lateral bending moments are appliedcontinuously at a constant rate of 1.7 degrees/second by stepper motorsintegrated in the gimbal of the spine tester. Two precycles are appliedto minimize the effect of the viscous component in the viscoelasticresponse, and data are collected on the third cyle. Range of motion,neutral zone, and two stiffness parameters are determined from theresulting load-deformation curves.

Histology/Histomorphometry

Eight specimens of each group are evaluated histologically after two,four or eight weeks postoperatively. After radiographic analysis, thespines are fixed in 10% formalin-solution. Cross sections of eitherspecimen are obtained to evaluate bony fusion, cellular reactions,biocompatibility, and signs of cement-integration/degradation.Qualitative histologic assessment of the fusion mass at the operativesite are made for the presence of giant cells, inflammatory cells, orfibrous responses where the implanted materials may have beenencapsulated. In addition, the osteoid found within the trabecularfusion mass and the amount of trabecular bone are assessed.Histomorphometric variables, such as the percentage of osteoid, osteoidthickness, number of osteoblasts per millimeter bone surface, and numberof osteoclasts per millimeter bone surface are determined.

Fluorochrome Labeling

Eight animals are subjected to intravenuous application of 90 milligramsof xylenol orange per kilogram of body weight two weeks postoperatively,10 milligrams of calcein green per kilogram of body weight four weekspostoperatively, and 25 milligrams of doxycyclinhyclate yellow perkilogram of body weight six weeks postoperatively. This regimen followsthe method published by Rahn and Perren. See, e.g., Rahn et al., StainTechnology, 46, pp. 125-129 (1971); Rahn et al., Akt Traumatol, 10, pp.109-115 (1980). Fluorochrome sequential analysis is then performed byFluorescence microscopy on the specimens under UV light for qualitativeand quantitative dynamic evaluation.

EXAMPLE 11 Repair of Osteochondral Defects in Dogs

A total of 12 adult male dogs are utilized. Bilateral osteochondraldefects, 5.0 mm in diameter and 6 mm deep, penetrating the subchondralbone, are created in the central load bearing region of each medialfemoral condyle. In 6 animals, the right defects will receive the highdose OP-1 encapsulated in PLGA. The left limb of all animals willreceive the collagen matrix plus CMC to serve as a control. Theremaining 6 dogs receive a low dose OP-1 encapsulated in PLGA on theright side and a control on the left side. The animals are sacrificed at16 weeks post-implantation. At sacrifice, the distal femurs areretrieved en bloc and the defect sites are evaluated histologically andgrossly based on the scheme of Moran et al., J. Bone Joint Surg. 74B,pp. 659-667 (1992), which is incorporated herein by reference.

Using standard aseptic techniques, surgery is performed underisofluorane gas anesthesia and the animals are monitored byelectrocardiogram and heart rate monitors. Pre-surgical medication isadministered approximately 20-30 minutes prior to anesthesia induction.The presurgical medication will consist of butorphanol tartrate (0.05mg/kg body weight). Anesthesia is administered by intravenous injectionof sodium pentothal (17.5 mg/kg body weight). Following induction, anendotracheal tube is placed and anesthesia is maintained by isofluoraneinhalation. Surgery is performed by making a medial parapatellarincision approximately 4 cm in length. The patella is retractedlaterally to expose the femoral condyle. In the right medial condyle, a5.0 mm diameter defect extending through the cartilage layer andpenetrating the subchondral bone to a depth of 6 mm is created with aspecially designed or modified 5.0 mm drill bit. After copiousirrigation with saline to remove bone debris and spilled marrow cells,the appropriate concentration of OP-1 encapsulated in PLGA is carefullypacked into each defect site with a blunt probe and by hand. Asufficient amount of OP-1 is placed within the defect so that it willflush with the articulating surface. While protecting the implantedmaterial, the joint is irrigated to remove any implant not placed withinthe defects. The joint capsule and soft-tissues are then meticulouslyclosed in layers with resorbable suture. The procedure is repeated onthe contralateral side with placement of a control.

Butorphanol tartrate (0.05 mg/kg body weight) is administeredsubcutaneously as required. Animals are administered intramuscularantibiotics for four days post-surgery and routine anterior-posteriorradiographs are taken immediately after surgery to insure propersurgical placement. Animals are kept in 3×4 feet recovery cages untilthe animal is able to tolerate weight bearing. Then, the animals aretransferred to runs and allowed unrestricted motion.

Radiographs of the hindlimbs are obtained preoperatively, immediatelypostoperative, and at 16 weeks (sacrifice). The preoperative radiographsare used to assure that no pre-existing abnormalities are present and toverify skeletal maturity. Postoperative radiographs are used to assessdefect placement. Sacrifice radiographs are used to assess the rate ofhealing and restoration of the subchondral bone and the articulatingsurface. Radiographs are obtained within one week of the evaluationdate.

At the appropriate time, animals are sacrificed using an intravenousbarbiturate overdose. The distal femurs are immediately harvested enbloc and stored in saline soaked towels, placed in plastic bags labeledwith the animal number, right or left designation, and any othernecessary identifiers. High power photographs of the defect sites aretaken and carefully labeled. Prior to sacrifice venous blood is drawnfor routine blood count with cell differential. Soft tissues aremeticulously dissected away from the defect site. The proximal end ofthe femur is removed. All specimens are prepared for histologicevaluation immediately after gross grading and photography. On a watercooled diamond saw each defect site is isolated.

The gross appearance of the defect sites and repair tissue is gradedbased upon the study of Moran et al., supra. Points are apportionedaccording to the presence of intra-articular adhesions, restoration ofthe articular surface, cartilage erosion and appearance.

The individual specimens are fixed by immersion in 4% paraformaldehydesolution and prepared for decalcified histologic processing. Threesections from three levels are cut from each block. Levels 1 and 3 areclosest to the defect perimeter. Level 2 is located at the defectcenter. Three sections from each level may be stained with toluidineblue and Safranin 0 and fast green. Sections are graded based upon thescheme of Moran et al., supra. This analysis apportions points basedupon the nature of the repair tissue, structural characteristics, andcellular changes. Descriptive statistics are calculated for gross andhistologic parameters.

While we have described a number of embodiments of this invention, it isapparent that our basic constructions may be altered to provide otherembodiments which utilize the products and processes of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims, rather than by the specificembodiments which have been presented by way of example.

1-69. (canceled)
 70. A method of inducing bone formation in a mammalscomprising the step of implanting at a bone defect site of said mammal acomposition comprising porous β-tricalcium phosphate (β-TCP) granulesthat have a particle size of 0.1-2 mm and that comprise a multiplicityof pores having a pore diameter size of 20-500 μm and being singleseparate voids partitioned by walls and being not interconnected. 71.The method of claim 70, wherein the composition further comprises abioactive agent.
 72. The method of claim 71, wherein the bioactive agentis a bone morphogenic protein.
 73. The method of claim 70, wherein thecomposition further comprises a binder. 74-85. (canceled)
 86. The methodof claim 73, wherein the binder is carboxy methylcellulose sodium. 87.The method of claim 70, wherein the porous β-TCP granules have aparticle size of 0.5-1 mm.
 88. The method of claim 71, wherein the porediameter size is 50-125 μm.